Anti-PD1 Antibodies and their Use as Therapeutics and Diagnostics

ABSTRACT

Provided are antibodies that specifically bind to Programmed Death-1 (PD1, Pdcd-1, or CD279) and inhibit PD1-mediated cellular signaling and activities in immune cells, antibodies binding to a set of amino acid residues required for its ligand binding, and uses of these antibodies to treat or diagnose cancer, infectious diseases or other pathological disorders modulated by PD1-mediated functions.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation of PCT/CN2013/083467, filed Sep. 13,2013 and a division of U.S. Ser. No. 14/194,797, filed Mar. 02, 2014(now US), which is a division of U.S. Ser. No. 14/076,214, filed Nov.10, 2013 (now U.S. Pat. No. 8,735,553).

INTRODUCTION

Programmed Death-1 (PD-1, also termed as CD279) is a 55 KD receptorprotein related to CD28/CTLA4 co-stimulatory/inhibitory receptor family(Blank et al., 2005 Cancer Immunol Immunother 54:307-314). The genes andcDNAs coding for PD-1 were cloned and characterized in mouse and human(Ishida et al., 1992 EMBO J 11:3887-3395; Shinohara et al., 1994Genomics 23:704-706). The full length PD-1 contains 288 amino acidresidues (NCBI accession number: NP_(—)005009). Its extracellular domainconsists of amino acid residues 1-167, and the cytoplasmic C-terminaltail comprises residues 191-288, which has two hypotheticalimmune-regulatory motifs, an immunoreceptor tyrosine-based inhibitorymotif (ITIM; Vivier et al., 1997 Immunol Today 18:286-291) and animmunoreceptor tyrosine switch motif (ITSM; Chemnitz et al., 2004 JImmunol 173:945-954).

To date, two sequence-related ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC),have been identified to specifically interact with PD-1, inducingintracellular signal transduction that inhibits CD3 and CD28 mediatedT-cell activation (Riley, 2009 Immunol Rev 229:114-125), which, in turn,attenuates T-cell activities, for example, reduction of cellproliferation, IL-2 and IFN-γ secretion, as well as other growth factorand cytokine secretion.

Expression of PD-1 was frequently found in immune cells such as T-cells,B-cells, monocytes and natural killer (NK) cells. It was rarelyexpressed in other human tissues, such as muscle, epithelium, neuronaltissues, etc. Furthermore, high level of PD-1 expression is oftenassociated with activation of immune cells. For example, when humanT-cell line, Jurkat, was activated by phytohaemagglutinin (PHA) orphorbol ester (12-O-tetradecanoylphorbol-13-acetate, or TPA), theexpression of PD-1 was up-regulated visible in Western Blot (Vibharka etal., 1997 Exp Cell Res 232:25-28). The same phenomenon was observed instimulated murine T- and B-lymphocytes and in primary human CD4⁺ T-cellsupon stimulation by anti-CD3 antibody (Agata et al., 1996 Int Immunol8:765-772; Bennett et al., 2003 J Immunol 170:711-118). The increase ofPD-1 expression following stimulation of T effector cells redirects theactivated T-effector cells towards exhaustion and reduced immuneactivities. Therefore, PD-1 mediated inhibitory signal plays animportant role in immune tolerance (Bour-Jordan et al., 2011 Immunol Rev241:180-205).

Increase of PD-1 expression in tumor-infiltrating lymphocytes (TILs) andPD-1 ligand expression in tumor cells were reported in varieties ofcancers involved in different types of tissues and organs such as lung(Konishi et al., 2004 Clin Cancer Res 10:5094-5100), liver (Shi et al.,2008 Int J Cancer 128:887-896; Gao et al., 2009 Clin Cancer Res15:971-979), stomach (Wu et al., 2006 Acta Histochem 108:19-24), kidney(Thompson et al., 2004 Proc Natl Acad Sci 101:17174-17179; Thompson etal., 2007 Clin Cancer Res 13:1757-1761), breast (Ghebeh et al., 2006Neoplasia 8:190-198), ovary (Hamanishi et al. 2007 Proc Natl Acad Sci104:3360-3365), pancreas (Nomi et al., 2007 Clin Cancer Res13:2151-2157), melanocytes (Hino et al., 2010 Cancer 116:1757-1766) andesophagus (Ohigashi et al., 2005 Clin Cancer Res 11:2947-2953). Morefrequently, the increased expression of PD-1 and PD-L1 in those cancersis associated with poor prognosis of patient survival outcome.Transgenic mice with PD-1 gene knockout inhibiting xenograft cancer cellgrowth further elucidated the significance of PD-1 signaling in themodulation of immune system for cancer eradication or tolerance (Zhanget al., 2009 Blood 114:1545-1552).

Not only does up-regulation of PD-1 signaling leads to immune toleranceto cancerous growth, but also to viral infection and expansion in human.The prevalent liver infection viruses, HBV and HCV, induceoverexpression of PD-1 ligands in hepatocytes and activate PD-1signaling in T-effector cells, resulting in T-cell exhaustion andtolerance to the viral infection (Boni et al., 2007 J Virol81:4215-4225; Golden-Mason et al., 2008 J Immunol 180:3637-3641).Likewise, HIV infection frequently evades human immune system by similarmechanisms. Therapeutic modulation of PD-1 signaling by antagonistmolecules may revert immune cells from tolerance, and reactivated toeradicate cancer and chronic viral infection (Blank et al., 2005 CancerImmunol Immunother 54:307-314; Okazaki et al., 2007 Int Immunol19:813-824).

SUMMARY OF THE INVENTION

The invention provides methods and compositions for immune-inhibition ofPD-1. In one aspect, the invention provides an antibody antigen bindingdomain which specifically binds human PD-1, and comprises acomplementarity determining region (CDR) having a sequence selected fromSEQ ID NOS 11-22, 31-42 and 59-63.

The CDRs are amenable to recombination into heavy chain variable region(Vh) and light chain variable regions (Vk) which comprise (CDR-H1,CDR-H2 and CDR-H3) and (CDR-L1, CDR-L2 and CDR-L3) sequences,respectively and retain PD-1-specific binding and/or functionality.

In particular embodiments, the domain comprises a heavy chain variableregion (Vh) or a light chain variable region (Vk) comprising:

a) CDR-H1 (SEQ ID NO: 11, 17, 31, or 37), b) CDR-H2 (SEQ ID NO: 12, 18,32, or 38), c) CDR-H3 (SEQ ID NO: 13, 18, 33, or 39); d) CDR-L1 (SEQ IDNO: 14, 20, 34, or 40), e) CDR-L2 (SEQ ID NO: 15, 21, 35, or 41), or f)CDR-L3 (SEQ ID NO: 16, 22, 36, or 42).

In particular embodiments, the domain comprises a heavy chain variableregion (Vh) and/or a light chain variable region (Vk) comprising:

a) mu317 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOS: 11-13); CDR-L1, CDR-L2and CDR-L3 (SEQ ID NOS: 14-16); b) mu326 CDR-H1, CDR-H2 and CDR-H3 (SEQID NOS: 17-19); CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOS: 20-22); c)317-4B6 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOS: 31-33); CDR-L1, CDR-L2and CDR-L3 (SEQ ID NOS: 34-36); d) 326-4A3 CDR-H1, CDR-H2 and CDR-H3(SEQ ID NOS: 37-39); CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOS: 40-42); e)317-1 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOS: 11, 59, 13); CDR-L1, CDR-L2and CDR-L3 (SEQ ID NOS: 14-16); f) 317-4B2 CDR-H1, CDR-H2 and CDR-H3(SEQ ID NOS: 11, 60, 13); CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOS: 61, 15,16); g) 317-4B5 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOS: 11, 60, 13);CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOS: 61, 15, 16); h) 317-4B6 CDR-H1,CDR-H2 and CDR-H3 (SEQ ID NOS: 11, 32, 13); CDR-L1, CDR-L2 and CDR-L3(SEQ ID NOS: 61, 15, 16); i) 326-1 CDR-H1, CDR-H2 and CDR-H3 (SEQ IDNOS: 17, 62, 19); CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOS: 20-22); j)326-3B1 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOS: 17, 62, 19); CDR-L1,CDR-L2 and CDR-L3 (SEQ ID NOS: 20-22); k) 326-3G1 CDR-H1, CDR-H2 andCDR-H3 (SEQ ID NOS: 17, 62, 19); or CDR-L1, CDR-L2 and CDR-L3 (SEQ IDNOS: 20-22).

In particular embodiments, the domain comprises a heavy chain variableregion (Vh) and a light chain variable region (Vk) comprising:

(a) CDR-H1 (SEQ ID NO 31), CDR-H2 (SEQ ID NO 12, 32, 59 or 60) andCDR-H3 (SEQ ID NO 33),

-   -   CDR-L1 (SEQ ID NO 14, 34 or 61), CDR-L2 (SEQ ID NO 35) and        CDR-L3 (SEQ ID NO 36); or

(b) CDR-H1 (SEQ ID NO 37), CDR-H2 (SEQ ID NO 18, 38 or 62) and CDR-H3(SEQ ID NO 39),

-   -   CDR-L1 (SEQ ID NO 40), CDR-L2 (SEQ ID NO 41) and CDR-L3 (SEQ ID        NO 42).

In particular embodiments, the domain comprises a heavy chain variableregion (Vh) or a light chain variable region (Vk) comprising:

a) mu317 (SEQ ID NOS: 4 or 6); b) mu326 (SEQ ID NOS: 8 or 10); c)317-4B6 (SEQ ID NOS: 24 or 26); d) 326-4A3 (SEQ ID NOS: 28 or 30); e)317-4B2 (SEQ ID NOS: 43 or 44); f) 317-4B5 (SEQ ID NOS: 45 or 46); g)317-1 (SEQ ID NOS: 48 or 50); h) 326-3B1 (SEQ ID NOS: 51 or 52); i)326-3G1 (SEQ ID NOS: 53 or 54); j) 326-1 (SEQ ID NOS: 56 or 58); k)317-3A1 (SEQ ID NOS: 64); l) 317-3C1 (SEQ ID NOS: 65); m) 317-3E1 (SEQID NOS: 66); n) 317-3F1 (SEQ ID NOS: 67); o) 317-3G1 (SEQ ID NOS: 68);p) 317-3H1 (SEQ ID NOS: 69); q) 317-3I1 (SEQ ID NOS: 70); r) 317-4B1(SEQ ID NOS: 71); s) 317-4B3 (SEQ ID NOS: 72); t) 317-4B4 (SEQ ID NOS:73); u) 317-4A2 (SEQ ID NOS: 74); v) 326-3A1 (SEQ ID NOS: 75); w)326-3C1 (SEQ ID NOS: 76); x) 326-3D1 (SEQ ID NOS: 77); y) 326-3E1 (SEQID NOS: 78); z) 326-3F1 (SEQ ID NOS: 79); aa) 326-3B N55D (SEQ ID NOS:80); ab) 326-4A1 (SEQ ID NOS: 81); or ac) 326-4A2 (SEQ ID NOS: 82).

In particular embodiments, the domain comprises a heavy chain variableregion (Vh) and a light chain variable region (Vk) comprising:

a) mu317 (SEQ ID NOS: 4 and 6); b) mu326 (SEQ ID NOS: 8 and 10); c)317-4B6 (SEQ ID NOS: 24 and 26); d) 326-4A3 (SEQ ID NOS: 28 and 30); e)317-4B2 (SEQ ID NOS: 43 and 44); f) 317-4B5 (SEQ ID NOS: 45 and 46); g)317-1 (SEQ ID NOS: 48 and 50); h) 326-3B1 (SEQ ID NOS: 51 and 52); i)326-3G1 (SEQ ID NOS: 53 and 54); j) 326-1 (SEQ ID NOS: 56 and 58); k)317-3A1 (SEQ ID NOS: 64 and 26); l) 317-3C1 (SEQ ID NOS: 65 and 26); m)317-3E1 (SEQ ID NOS: 66 and 26); n) 317-3F1 (SEQ ID NOS: 67 and 26); o)317-3G1 (SEQ ID NOS: 68 and 26); p) 317-3H1 (SEQ ID NOS: 69 and 26); q)317-3I1 (SEQ ID NOS: 70 and 26); r) 317-4B1 (SEQ ID NOS: 71 and 26); s)317-4B3 (SEQ ID NOS: 72 and 26); t) 317-4B4 (SEQ ID NOS: 73 and 26); u)317-4A2 (SEQ ID NOS: 74 and 26); v) 326-3A1 (SEQ ID NOS: 75 and 30); w)326-3C1 (SEQ ID NOS: 76 and 30); x) 326-3D1 (SEQ ID NOS: 77 and 30); y)326-3E1 (SEQ ID NOS: 78 and 30); z) 326-3F1 (SEQ ID NOS: 79 and 30); aa)326-3B N55D (SEQ ID NOS: 80 and 30); ab) 326-4A1 (SEQ ID NOS: 28 and81); or ac) 326-4A2 (SEQ ID NOS: 28 and 82).

In particular embodiments, the domain specifically binds PD1 residues:(a) K45 and I93 (AA numbering based on 2008 PNAS, 105:10483; equivalentto K58 and 1106 in SEQ ID NO 2); or (b) I93, L95 and P97(AA numberingbased on 2008 PNAS, 105:10483; equivalent to I106, L108 and P110 in SEQID NO 2).

In particular embodiments, the domain induces IL-2 release in HuT78/PD-1cells co-cultured with HEK293/OS8/PD-L1 cells or with EK293/OS8/PD-L2cells, and/or inhibits IL-2 secretion in HuT78/P3Z cells co-culturedwith HEK293/PD-L1 cells or with HEK293/PD-L2 cells.

The invention also provides an antibody IgG4 heavy chain effector orconstant domain comprising any of SEQ ID NO:83-88, particularly SEQ IDNO 87 or 88.

The invention also provides antibodies, F(ab) or F(ab)2 comprising asubject PD-1 binding domain.

The invention also provides antibodies comprising a subject PD-1 bindingdomain and a IgG4 heavy chain effector or constant domain comprising anyof SEQ ID NO:83-88, particularly SEQ ID NO 87 or 88.

The invention also provides a polynucleotide encoding a subject PD-1binding domain, particularly cDNA sequences.

The invention provides methods of using the subject domains byadministering the domain to a person determined to have cancer or aviral infection or to otherwise be in need of PD-1 antagonism.

The invention also provides fusion proteins comprising: (a) a singlechain variable fragment (scFv) of an anti-human CD3 mAb OKT3 fused tothe C-terminal domain (113-220) of mouse CD8α (SEQ ID NO:89); or (b) theextracellular and transmembrane domains of human PD-1 fused to thecytoplasmic domain of human CD3ζ chain (SEQ ID NO: 90).

The invention also provides methods of using the subject fusionproteins, comprising assaying, screening or selecting anti-PD-1antibodies with a cell line expressing the fusion protein.

BREIF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic presentation of PD-1/Fc (top) and PD-1/His (bottom).ECD: extracellular domain. L: linker. H: His tag. Fc: γ4Fc fragment fromhuman IgG4. N: N-terminus C: C-terminus

FIG. 2. Dose-dependent reaction curves of murine mAbs binding to humanPD-1 in ELISA. The murine mAbs were indicated at top—left corner of eachfigure. MAb 317 and 517 share high degree of homology the variableregion of heavy and light chains. The binding signal strength wasindicated by direct OD₄₅₀ readings. The antigen, PD-1/His, was coated atincreasing concentrations up to 70 nanograms per well in a volume of 50microliters. The method was described in Example 1.

FIG. 3. Dose-dependent reaction curve of murine mAbs binding to humanPD-1 expressed on live cells by FACS analyses. Murine antibody codes andEC₅₀ were indicated on each panel. MFI stands for mean fluorescenceintensity. HuT78/PD-1 cells were suspended in 96-well plate at 5×10⁴cells per well for FACS. PD-1 mAbs binding to the cell surface targetand FACS detection were performed as described in Example 1.

FIG. 4. Schematic presentation of the cell co-culture systems used forassaying functional activities of anti-PD-1 mAbs. T-cells (either CD4⁺or CD8⁺) represent HuT78/PD-1 or primary T-cells in PBMCs. TCR: T-cellreceptor. N: nucleus. C: cytoplasm

FIG. 5. Dose-dependent reaction curve of murine mAb-induced IL-2secretion in HuT78/PD-1 cells co-cultured with HEK293/OS8/PD-L1 cells.Baseline: Average IL-2 release induced by mIgGs at all testedconcentrations. Top line: Highest IL-2 release based on regressioncalculation by Prizm Software.

FIG. 6. (A) Histograms showing IFN-γ secretion induced by anti-PD-1 mAbsin PBMCs (Donor-19) co-cultured with cell line HEK293/OS8/PD-L1. (B)Histograms showing IFN-γ secretion induced by anti-PD-1 mAbs in PBMCs(Donor-20) co-cultured with cell line HEK293/OS8/PD-L1.

FIG. 7. (A) and (B) ADCC activities of anti-PD-1 mAbs by co-culture ofeffector cells (NK92MI/PD-1) and target cells (HuT78/PD-1). Means werecalculated from two data points of the representative experiments. ThemAbs were added to concentration of 10 μm/ml. Experiment performed asdescribed in Example 9.

FIG. 8. Mapping the binding epitopes of anti-PD-1 mAbs by ELISA(up-panel) and Western Blot (lower panel). Conditioned media containingWT or Mt PD-1 were used to assess binding activity by ELISA and WesternBlot. **indicates the AA residues to which the mAb binding activityreduced to 25-50% of WT PD-1. *** indicates the AA residues to which themAb binding activity reduced below 25% of WT PD-1.

FIG. 9. IFN-γ release induced by humanized anti-PD-1 mAbs in primaryhuman PBMCs from different healthy donors, which were co-cultured withHEK293/OS8/PD-L1 cells.

FIG. 10. Cytotoxicity of NK92MI/PD-1 cells enhanced by humanizedanti-PD-1 mAbs, hu317 (A) and hu326 (B). The target lung cancer cells,SK-MES-1/PD-L1, were co-cultured with the effector cells at the (T to E)ratio of 1 to 2, and assayed as described in Example 12.

FIG. 11. Individual tumor growth curves in three treatment groups,vehicle (PBS), human IgGs (huIgGs) and anti-PD-1 mAb (hu317-1/IgG4mt2).Each curve represents a tumor growth path, the tumor-bearing mice codedby numbers indicated on the right of each panel. Hep3B/OS8/PD-L1 cells(established from hepatocellular carcinoma line Hep3B) were seeded atDay 1, PBMCs were implanted at Day 15 and three doses of hu317-1/IgG4mt2were injected at Day 18, 28 and 38, respectively. Methods described inExample 12.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

PD-1 initiates inhibitory signaling in immune cells when engaged by itsligands, PD-L1 or PD-L2. In the cases of cancer outgrowth and viralinfection, the activation of PD-1 signaling promotes immune tolerance,leading to the cancers or virus-infected cells escaping from immunesurveillance and cancer metastasis or viral load increase Inhibition ofPD-1 mediated cellular signaling by therapeutic agents can activateimmune cells including T-cells, B-cells and NK cells, and thereforeenhance immune cell functions inhibiting cancer cell growth or viralinfection, and restore immune surveillance and immune memory function totreat such human diseases.

The invention provides antibodies whose functions are antagonistic tothe ligand-induced and PD-1-mediated cellular signaling in immune cells.Murine anti-PD-1 antibodies were humanized to a high degree ofsimilarity to human antibodies in the framework regions. The fullantibodies made in the modified human IgG4 variant format have a uniqueset of features in the aspects of effector functions and physicochemicalproperties. The disclosed anti-PD-1 antibodies are suitable fortherapeutic uses in cancer treatment, controlling viral infections andother human diseases that are mechanistically involved in exacerbatedimmune tolerance.

Definitions

Unless the context indicates otherwise, the term “antibody” is used inthe broadest sense and specifically covers antibodies (including fulllength monoclonal antibodies) and antibody fragments so long as theyrecognize PD-1. An antibody molecule is usually monospecific, but mayalso be described as idiospecific, heterospecific, or polyspecific.Antibody molecules bind by means of specific binding sites to specificantigenic determinants or epitopes on antigens. “Antibody fragments”comprise a portion of a full length antibody, generally the antigenbinding or variable region thereof. Examples of antibody fragmentsinclude Fab, Fab', F(ab').sub.2, and Fv fragments; diabodies; linearantibodies; single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

Natural and engineered antibody structures are well known in the art,e.g. Strohl et al., Therapeutic antibody engineering: Current and futureadvances driving the strongest growth area in the pharmaceuticalindustry, Woodhead Publishing Series in Biomedicine No. 11, October2012; Holliger et al. Nature Biotechnol 23, 1126-1136 (2005); Chames etal. Br J Pharmacol. 2009 May; 157(2): 220-233.

Monoclonal antibodies (MAbs) may be obtained by methods known to thoseskilled in the art. See, for example Kohler et al (1975); U.S. Pat. No.4,376,110; Ausubel et al (1987-1999); Harlow et al (1988); and Colliganet al (1993). The mAbs of the invention may be of any immunoglobulinclass including IgG, IgM, IgE, IgA, and any subclass thereof. Ahybridoma producing a mAb may be cultivated in vitro or in vivo. Hightiters of mAbs can be obtained in in vivo production where cells fromthe individual hybridomas are injected intraperitoneally into mice, suchas pristine-primed Balb/c mice to produce ascites fluid containing highconcentrations of the desired mAbs. MAbs of isotype IgM or IgG may bepurified from such ascites fluids, or from culture supernatants, usingcolumn chromatography methods well known to those of skill in the art.

An “isolated polynucleotide” refers to a polynucleotide segment orfragment which has been separated from sequences which flank it in anaturally occurring state, e.g., a DNA fragment which has been removedfrom the sequences which are normally adjacent to the fragment, e.g.,the sequences adjacent to the fragment in a genome in which it naturallyoccurs. The term therefore includes, for example, a recombinant DNAwhich is incorporated into a vector, into an autonomously replicatingplasmid or virus, or into the genomic DNA of a prokaryote or eukaryote,or which exists as a separate molecule (e.g., as a cDNA or a genomic orcDNA fragment produced by PCR or restriction enzyme digestion)independent of other sequences. It also includes a recombinant DNA,which is part of a hybrid gene encoding additional polypeptide sequence.

A “construct” means any recombinant polynucleotide molecule such as aplasmid, cosmid, virus, autonomously replicating polynucleotidemolecule, phage, or linear or circular single-stranded ordouble-stranded DNA or RNA polynucleotide molecule, derived from anysource, capable of genomic integration or autonomous replication,comprising a polynucleotide molecule where one or more polynucleotidemolecule has been linked in a functionally operative manner, i.e.operably linked. A recombinant construct will typically comprise thepolynucleotides of the invention operably linked to transcriptionalinitiation regulatory sequences that will direct the transcription ofthe polynucleotide in the intended host cell. Both heterologous andnon-heterologous (i.e., endogenous) promoters can be employed to directexpression of the nucleic acids of the invention.

A “vector” refers any recombinant polynucleotide construct that may beused for the purpose of transformation, i.e. the introduction ofheterologous DNA into a host cell. One type of vector is a “plasmid”,which refers to a circular double stranded DNA loop into whichadditional DNA segments can be ligated. Another type of vector is aviral vector, wherein additional DNA segments can be ligated into theviral genome. Certain vectors are capable of autonomous replication in ahost cell into which they are introduced (e.g., bacterial vectors havinga bacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”.

An “expression vector” as used herein refers to a nucleic acid moleculecapable of replication and expressing a gene of interest whentransformed, transfected or transduced into a host cell. The expressionvectors comprise one or more phenotypic selectable markers and an originof replication to ensure maintenance of the vector and to, if desired,provide amplification within the host. The expression vector furthercomprises a promoter to drive the expression of the polypeptide withinthe cells. Suitable expression vectors may be plasmids derived, forexample, from pBR322 or various pUC plasmids, which are commerciallyavailable. Other expression vectors may be derived from bacteriophage,phagemid, or cosmid expression vectors.

Additional Embodiments of the Invention

In specific embodiments the invention provides mouse monoclonalantibodies identified from screening murine hybridoma clones asdisclosed herein.

In other embodiments the invention provides compositions of thefollowing polynucleotide and protein sequences:

a) The cDNA sequence, SEQ ID NO 3, encoding the heavy chain variableregion of murine mAb 317;

b) The protein sequence of the heavy chain variable region of murine mAb317 or mu317_Vh (SEQ ID NO 4);

c) The cDNA sequence, SEQ ID NO 5, encoding the light chain variableregion of murine mAb 317;

d) The protein sequence of the light chain variable region of murine mAb317 or mu317_Vk (SEQ ID NO 6);

e) The cDNA sequence, SEQ ID NO 7, encoding the heavy chain variableregion of murine mAb 326;

f) The protein sequence of the heavy chain variable region of murine mAb326 or mu326_Vh (SEQ ID NO 8);

g) The cDNA sequence, SEQ ID NO 9, encoding the light chain variableregion of murine mAb 326;

h) The protein sequence of the light chain variable region of murine mAb326 or mu326_Vk (SEQ ID NO 10).

In one aspect, the invention provides compositions comprising complementdeterminant region (CDR) sequences, which mediate binding to the targetantigens, PD-1, including the CDR sequences of mu317 and m326:

a) The CDR1 of mu317 heavy chain (mu317 H-CDR1) contains amino acidsequence of GFSLTSYGVH (SEQ ID NO 11);

b) The mu317 H-CDR2 contains amino acid sequence of VIWAGGSTNYNSALMS(SEQ ID NO 12);

c) The mu317 H-CDR3 contains amino acid sequence of ARAYGNYWYIDV (SEQ IDNO 13);

d) The CDR1 of mu317 light chain (mu317 L-CDR1) contains amino acidsequence of KASQSVSNDVA (SEQ ID NO 14);

e) The mu317 L-CDR2 contains amino acid sequence of YAFHRFT (SEQ ID NO15);

f) The mu317 L-CDR3 contains amino acid sequence of HQAYSSPYT (SEQ NO16);

g) The mu326 H-CDR1 contains amino acid sequence of GYTFTNYGMN (SEQ IDNO 17);

h) The mu326 H-CDR2 contains amino acid sequence of WINNNNGEPTYAEEFKG(SEQ ID NO 18);

i) The mu326 H-CDR3 contains amino acid sequence of ARDVMDY (SEQ ID NO19);

j) The mu326 L-CDR1 contains amino acid sequence of RASESVDNYGYSFMH (SEQID NO 20);

k) The mu326 L-CDR2 contains amino acid sequence of RASNLES (SEQ ID NO21);

l) The mu326 L-CDR3 contains amino acid sequence of QQSKEYPT (SEQ ID NO22).

In another embodiment, the invention provides compositions comprisingthe sequences of the humanization monoclonal antibodies emanated frommurine mAbs mu317 and mu326, incuding:

a) The humanization mAb hu317-4B6 comprises protein sequence of heavychain variable region (Vh) as SEQ ID NO 24, which is encoded by

b) the cDNA of hu317-4B6_Vh (SEQ ID NO 23);

c) The humanization mAb hu317-4B6 also comprises protein sequence oflight chain variable region (Vk) as SEQ ID NO 26, which is encoded by

d) the cDNA of hu317-4B6 (SEQ ID NO 25);

e) he humanization mAb hu326-4A3 comprises protein sequence of Vh as SEQID NO 28, which is encoded by

f) the cDNA of hu326-4A3-Vh (SEQ ID NO 27);

g) The humanization mAb hu326-4A3 also comprises protein sequence of Vkas SEQ ID NO 30, which is encoded by

h) the cDNA of hu326-4A3_Vk (SEQ ID NO 29);

i) The protein sequences of hu317-4B2_Vh (SEQ ID NO 43) and hu317-4B2_Vk(SEQ ID NO 44);

j) The protein sequences of hu317-4B5_Vh (SEQ ID NO 45) and hu317-4B5_Vk(SEQ ID NO 46);

k) The protein sequence of hu317-1_Vh (SEQ ID NO 48) and the cDNAencoding for hu317-1_Vh (SEQ ID NO 47);

l) The protein sequence of hu317-1_Vk (SEQ ID NO 50) and the cDNAencoding for hu317-1_Vk (SEQ ID NO 49);

m) The protein sequences of hu326-3B1_Vh (SEQ ID NO 51) and hu326-3B1_Vk(SEQ ID NO 52);

n) The protein sequences of hu326-3G1_Vh (SEQ ID NO 53) and hu326-3G1_Vk(SEQ ID NO 54);

o) The protein sequence of hu326-1_Vh (SEQ ID NO 56) and the cDNAencoding for hu326-1_Vh (SEQ ID NO 55);

p) The protein sequence of hu326-1_Vk (SEQ ID NO 58) and the cDNAencoding for hu326-1_Vk (SEQ ID NO 57);

q) The protein sequences of other humanization mAbs emanated from mu317(SEQ ID NO 63-74);

r) The protein sequences of other humanization mAbs emanated from mu326(SEQ ID NO 75-82);

In one aspect, the invention provides compositions comprising the CDRsequences of the humanization monoclonal antibodies. The CDRs may beshared among the same series of humanization mAbs, such as hu317 orhu326 (see Table 15-16). Non-redundant CDRs are listed below:

a) H-CDR1 sequence of GFSLTSYGVH (SEQ ID NO 31), shared throughouthumanization mAbs hu317 and mu317 in the heavy chains;

b) H-CDR3 sequence of ARAYGNYWYIDV (SEQ ID NO 33), shared throughouthumanization mAbs hu317 and mu317 in the heavy chains;

c) L-CDR1 sequence of KSSESVSNDVA (SEQ ID NO 34), shared throughouthumanization mAbs hu317-4B2, hu317-4B5 and hu317-4B6 in the lightchains;

d) L-CDR2 sequence of YAFHRFT (SEQ ID NO 35), shared throughouthumanization mAbs hu317 and mu317 in the light chains;

e) L-CDR3 sequence of HQAYSSPYT (SEQ ID NO 36), shared throughouthumanization mAbs hu317 and mu317 in the light chains;

f) H-CDR2 sequence of VIYADGSTNYNPSLKS (SEQ ID NO 32) in hu317-4B6_Vh;

g) H-CDR2 sequence of VIYAGGSTNYNPSLKS (SEQ ID NO 60) in hu317-4B2_Vhand hu317-4B5_Vh;

h) H-CDR2 sequence of VIWAGGSTNYNPSLKS (SEQ ID NO 59) in hu317-1_Vh;

i) L-CDR1 sequence of KASQSVSNDVA (SEQ ID NO 11) in hu317-1_Vk;

j) H-CDR1 sequence of GYTFTNYGMN (SEQ ID NO 37), shared throughouthumanization mAbs hu326 and mu326 in the heavy chains;

k) H-CDR3 sequence of ARDVMDY (SEQ ID NO 39), shared throughouthumanization mAbs hu326 and mu326 in the heavy chains;

l) L-CDR1 sequence of RASESVDNYGYSFMH (SEQ ID NO 40), shared throughouthumanization mAbs hu326 and mu326 in the light chains;

m) L-CDR2 sequence of RASNLES (SEQ ID NO 41), shared throughouthumanization mAbs hu326 and mu326 in the light chains;

n) L-CDR3 sequence of QQSKEYPT (SEQ ID NO 42), shared throughouthumanization mAbs hu326 and mu326 in the light chains;

o) H-CDR2 sequence of WINNNNAEPTYAQDFRG (SEQ ID NO 38) inhu326_(—)4A3_Vh;

p) H-CDR2 sequence of WINNNNGEPTYAQGFRG (SEQ ID NO 62) in the Vh ofhu326_(—)1 and other hu317 mAbs.

In another aspect, the invention provides particular binding epitopes ofthe humanized anti-PD-1 mAbs on the antigen, and functional use thereof.Six critical amino acid (AA) residues in PD-1 required for the ligandbinding were mutated individually, and mutant and wild-type PD-1proteins were used to assess the binding epitopes. The residue whosemutation significantly impaired the antibody binding is recognized as akey or significant binding epitope. Significant binding epitopes of mAbshu317-4B5 and hu317-4B6 are K45 and I93 (AA numbering based on 2008PNAS, 105:10483; equivalent to K58 and 1106 in SEQ ID NO 2); andsignificant binding epitopes of mAbs hu326-3B 1 and hu317-4A3 are I93,L95 and P97 (AA numbering based on 2008 PNAS, 105:10483; equivalent toI106, L108 and P110 in SEQ ID NO 2).

In a further aspect, the invention provides compositions comprising theconstant region sequences of recombinant human IgG4 variants, which maybe linked to the variable regions of the subject antibodies, includingthe humanized anti-PD-1 mAbs, which showed preferred effector functionsand physicochemical properties. The sequences are as follows:

The constant region sequence of IgG4mt10 (SEQ ID NO 88);

a) A reference sequence of IgG4mt1 (SEQ ID NO 83);

b) A reference sequence of IgG4mt2 (SEQ ID NO 84);

c) A reference sequence of IgG4mt6 (SEQ ID NO 85);

d) A reference sequence of IgG4mt8 (SEQ ID NO 86);

e) A reference sequence of IgG4mt9 (SEQ ID NO 87).

In another embodiment, the invention provides methods for assayinganti-PD-1 antibody functions, using a plasmid expressing the recombinantfusion protein, OS8, to generate stable cell lines, HEK293/OS8/PD-L1 orHEK293/OS8/PD-L2, which co-expresses OS8 (a T cell-activating molecule)and a PD-1 ligand. The cell lines were used to engage T-cells and PBMCsby co-culture to assess the functionality of anti-PD-1 mAbs (see Example3 and Example 4). Alternatively, another plasmid expressing therecombinant fusion protein, P3Z, was used to generate stable cell line,HuT78/P3Z, in which P3Z functions as molecular sensor and signaltransduction mediator. When P3Z is engaged by PD-1 ligand, it willtransmit intracellular signal to activate IL-2 release in the HuT78cells. The systems may be used to assess inhibitory effect of anti-PD-1mAbs (see Example 3).

In one aspect, the invention provides compositions comprising the aminoacid sequences of the recombinant fusion proteins as follows:

a) Protein sequence of OS8 (SEQ ID NO 89);

b) Protein sequence of P3Z (SEQ ID NO 90).

In another aspect, the invention provides methods of generating thestable cell lines that express the recombinant fusion proteins describedherein, and methods of using the system to quantitatively assay thefunctional activities of anti-PD-1 mAbs.

In another embodiment the invention provides polynucleotides encodingthe subject proteins. The polynucleotides may be operably linked to aheterologous transcription regulating sequence for expression, and maybe incorporated into vectors, cells, etc.

In another embodiment, the invention provides the murine anti-PD-1antibodies and humanized version anti-PD-1 antibodies, includinghu317-4B6, hu317-4B5, hu317-4B2, etc., and hu326-4A3, hu326-3B1,hu326-3G1, etc., having functions to suppress PD-1 mediated signaltransduction, and to activate immune cells, which trigger a cascade ofimmune responses including cytokine secretion and cytotoxicity towardstarget cells such as cancer cells, and such functional use of theantibodies.

In one aspect, the invention provides humanized anti-PD-1 antibodiesthat activate several types of immune cells that express PD-1, includinghuman T-cells, NK-cells and PBMCs, whose functions are to amplify theimmune response signals, to mobilize immune system and to act as immuneeffector cells for clearance of cancer cells and viral infections, andsuch functional use of the antibodies.

In another aspect, the humanized anti-PD-1 mAbs are used as therapeuticagents to treat human diseases that are involved in suppression ofimmune cells by PD-1 mediated intracellular signaling, leading todisease progression, particularly cancers and viral infections.

The compositions of the invention are useful for the treatment ofcancer, neurodegenerative and infectious, particularly viral, diseasesand other conditions in which inappropriate or detrimental expression ofthe human PD-1 and/or is a component of the etiology or pathology of thecondition. Hence, the invention provides methods for treating cancer orinhibiting tumor progression in a subject in need thereof with a subjectanti-PD-1 protein. The invention further provides the use of subjectpolynucleotides for the manufacture of a medicament for treating canceror inhibiting tumor progression in a subject.

The invention includes all combinations of the recited particularembodiments. Further embodiments and the full scope of applicability ofthe invention will become apparent from the detailed description givenhereinafter. However, it should be understood that the detaileddescription and specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only,since various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thisdetailed description. All publications, patents, and patent applicationscited herein, including citations therein, are hereby incorporated byreference in their entirety for all purposes.

EXAMPLES Example 1 Generation of Anti-PD-1 Monoclonal Antibody

Anti-PD-1 monoclonal antibodies (mAbs) were generated based onconventional hybridoma fusion technology (Kohler and Milstein 1976 Eur JImmunol 6:511-519; de St Groth and Sheidegger 1980, J Immunol Methods35:1-21; Mechetner 2007 Methods Mol Biol 378:1-13) with minormodifications. MAbs with high binding activities in enzyme-linkedimmunosorbent assay (ELISA) and fluorescence-activated cell sorting(FACS) assay were selected for further characterization

PD-1 Recombinant Protein for Immunization and Binding Assays

Expression plasmid containing full-length human PD-1 cDNA was obtainedfrom Origene (Cat. No. SC117011, NCBI Accession No: NM_(—)005018.1,Beijing, China). The extracellular domain consisting of amino acid (AA)1-168 of PD-1 (SEQ NO.1, SEQ NO.2) was PCR-amplified, and subcloned inpcDNA3.1-based expression vector (Invitrogen, Carlsbad, Calif., USA)with C-terminus fused either to a His₆ tag or to the γFc domain of humanIgG4 heavy chain, which resulted in two recombinant fusion proteinexpression plasmids, PD-1-EC/His and PD-1-EC/Fc (abbreviated as PD-1/Hisand PD-1/Fc). The schematic presentation of immunogen/antigen proteinswere shown in FIG. 1. For the recombinant fusion protein production,PD-1/His and PD-1/Fc plasmids were transiently transfected into 293-Fcells in 1-3 liters of medium (Invitrogen), and cultured for 5-7 days ina CO₂ incubator equipped with rotating shaker. The supernatantcontaining the recombinant protein was collected and cleared bycentrifugation at 15000 g for 30 minutes. PD-1/His was purified throughimmobilized metal affinity chromatography using Ni-Sepharose Fast Flow(Cat. No. 17531801, GE Lifesciences, Shanghai, China), followed by sizeexclusion chromatography using a HiLoad 16/60 Superdex 200 column (Cat.No. 17106901, GE Lifesciences, Shanghai, China). PD-1/Fc was purifiedusing a Protein G Sepharose Fast Flow column (Cat. No. 17061805, GELifesciences). Both PD-1/His and PD-1/Fc proteins were dialyzed againstphosphate buffered saline (PBS) and stored in −80° C. freezer in smallaliquots.

The cDNA coding for human PD-L1 was chemically synthesized by Genescript(Nanjing, China) based on the published sequence (NCBI Accession No.NM_(—)014143). The PD-L2 expression plasmid was purchased from Origene(Cat. No. SC 108873, NCBI Accession No. NM_(—)025239.2, Beijing, China).Both cDNAs were cloned in pcDNA3.1/Hygromycin (Cat. No. V870-20,Invitrogen), and pcDNA3.1/V5-His (Cat. No. V810-20, Invitrogen),respectively.

Stable Expression Cell Line

Stable cell lines expressing human PD-1, PD-L1 or PD-L2 were establishedby transfection of pcDNA3.1 plasmids containing PD-1, PD-L1 and PD-L2 toHUT78 (ATCC, Manassas, Va., USA) and HEK293 (ATCC), respectively, andfollowed by selection with medium containing 200 micrograms ofhygromycin (Cat. No. 10687-010, Invitrogen) or 1 mg of G418 (Sigma) permilliliter. Single clones were isolated by conventional method, eitherlimited dilution or picking up single colonies from culture-wellsurface. All clones were screened by Western blot and FACS analysisusing anti-PD-1, PD-L1 and PD-L2 antibodies (Cat. No. 12-9969, 17-5983,12-5888, eBioscience, San Diego, USA), respectively, and the topexpression clones were selected for FACS binding assay to screenhybridoma monoclonal antibodies, or used in functional assays.

Immunization, Hybridoma Fusion and Cloning

Eight to twelve week-old Balb/c mice (from BEIJING HFK BIOCSIENCE CO.,LTD, Beijing, China) were immunized subcutaneously with 100 ul ofadjuvant (Cat. No. KX0210041, KangBiQuan, Beijing, China) containing 5micrograms of PD-1/Fc. The immunization was conducted by two injectionsof the above immunogen with three weeks apart. Two weeks after the 2ndimmunization, the mice sera were evaluated for PD-1 binding by FACS(following sections). The mice with high anti-PD-1 antibody titers insera were selected and boosted intraperitoneally with 50 micrograms ofPD-1/Fc in the absence of any adjuvant. Three days after boosting, thesplenocytes were isolated and fused with the murine myeloma cell line,SP2/0 cells (ATCC), using standard techniques (Gefter, M. L. et al.,1977 Somat Cell Genet, 3:231-236).

Assess PD-1 Binding Activity of Antibodies by ELISA and FACS

The supernatants of hybridoma clones were initially screened byEnzyme-Linked Immuno-Sorbent Assay (ELISA) as described in “Flanagan, M.L. et al. 2007 Methods in Molecular Biology 378:33-52” with somemodifications. Briefly, 50-200 nanograms of PD-1/His or PD-1/Fc proteinin 50 microliters of phosphate buffered saline (PBS) were coated in96-well plate (Shenzhen JinCanHua Industry Co., Ltd, Shenzhen, China) onper well base. The HRP-linked anti-mouse IgG antibody (Cat. No. 7076S,Cell Signaling Technology, USA and Shanghai, China) and chemiluminescentreagent (Cat. No. PA107-01, TIANGEN, China) were used to detect anddevelop the ELISA signal, which were read out by a plate reader(PHREAstar FS, BMG LABTECH, Germany) at wavelength of 450 nm. TheELISA-positive antibody producer clones were further verified byfluorescence-activated cell sorting (FACS) using a conventional method.PD-1 stable expression cell lines, HuT78/PD-1 (10⁵ cells/well),described above, was stained with supernatants from anti-PD-1 hybridomasin V-bottom 96-well plates (Cat. No. 3897, Corning, USA and Shanghai,China). To block human Fc receptors, cells were pre-incubated with humanIgG (20 μg/ml) (Cat. No. H11296, LifeHolder, USA and Shanghai, China).PD-1 antibodies were detected with Dylight™ 649-labelled goat anti-mouseIgG antibody (Cat. No. 405312, Biolegend, San Diego, USA) and cellfluorescence was monitored using a flow cytometer (Guava easyCyte 8HT,Merck-Millipore, USA and Shanghai, China).

The conditioned media of hybridoma cells that showed positive signal inboth ELISA and FACS assay were subjected to functional assays toidentify antibodies with good functional activity in human immunecell-based assays (herein). The antibodies with positive functionalactivity were further subcloned and characterized.

Subcloning and Adaptation to Serum Free or Low Serum Medium

The positive hybridoma clones from primary screening through ELISA, FACSand functional assays were subcloned by the conventional method oflimited dilution. Each of the positive clones was plated out in a96-well plate, cultured in RPMI1640 medium (Cat. No. SH30809.01B,Hyclone, Shanghai, China) with 10% fetal bovine serum (FBS, Cat. No.SH30084.03, Hyclone, Beijing, China) in CO₂ incubator. Three subclonesfrom each limited dilution plate were selected and characterized by FACSand functional assays. The subclones selected through functional assayswere defined as monoclonal antibody. The top subclones were adapted forgrowth in the CDM4MAb medium (Cat. No. SH30801.02, Hyclone) with 1-3%FBS.

Expression and Purification of Monoclonal Antibodies

Either murine monoclonal antibody-producing hybridoma cells orrecombinant antibody plasmids-transfected 293-F cells (Cat. No. R79007,Invitrogen) were cultured in CDM4MAb medium (Cat. No. SH30801.02,Hyclone) or Freestyle293 Expression medium (Cat. No. 12338018,Invitrogen), respectively, in a CO₂ incubator at 37° C. for 5 to 7 days.The conditioned medium was collected through centrifugation at 10,000 gfor 30 minutes to remove all cells and cell debris, and filtratedthrough a 0.22 μm membrane before purification. Murine or recombinantantibodies were applied and bound to a Protein A column (Cat. No.17127901, GE Life Sciences) following the manufacturer's guidance,washed with PBS, eluted in the buffer containing 20 mM citrate, 150 mMNaCl, pH3.5. The eluted materials were neutralized with 1M Tris pH8.0,and usually contained antibodies of above 90% purity. The ProteinA-affinity purified antibodies were either dialyzed against PBS orfurther purified using a HiLoad 16/60 Superdex200 column (Cat. No.17531801, GE Life Sciences) to remove aggregates. Protein concentrationswere determined by measuring absorbance at 280 nm or by Bradford assay(Cat. No. 1856210, Thermo Scientific, Rockford, Ill., USA) using bovineIgG of defined concentration (Cat. No. 23212, Thermo Scientific) as thestandards. The purified antibodies were stored in aliquots in −80° C.freezer.

Example 2 Comparison of Binding Activities Among Anti-PD-1 Antibodies

Through screening thousands of hybridomal clones we identified some topmonoclonal antibodies (mAb), which bind to human PD-1 with highspecificity and strength. As shown in ELISA assay (FIG. 2), three of thetop antibodies elicited such binding strength and specificity. FACSanalysis results demonstrated the selected monoclonal antibodies bind tothe native PD-1 proteins expressed on cell surface. Murine mAb317(mu317), mu326 and mu150 showed concentration-dependent bindingactivity, and their binding EC₅₀ (Effective concentration at 50%activity) was significantly lower than that of the control mu55 (FIG.3).

Assess mAb Binding Affinity by Surface Plasmon Resonance (SPR)

The mAbs with high binding activities in ELISA and FACS, as well as withpotent functional activities in the cell-based assays (herein) wereexamined for their binding kinetic constant in real time bindingreactions. Murine anti-PD-1 mAbs were purified from hybridomasupernatants using protein A Flow column (Cat. No. 17531801, GE LifeSciences) followed by exclusion chromatography using a HiLoad 16/60Superdex200 column (Cat. No. 17106901, GE Life Sciences). The purifiedanti-PD-1 antibodies were concentrated to 0.5-1 mg/mL in PBS and storedin aliquots in −80° C. freezer.

For determining binding affinities of PD-1 mAbs, SPR measurements wereperformed in HBS-N buffer (10 mM HEPES pH 7.4, 0.15M NaCl, 3 mM EDTA,0.005% v/v surfactant P20, GE Healthcare) using the BIAcore™ T-200instrument (GE Life Sciences). Anti-mouse Fc CM5 biosensor chip (GEHealthcare) was generated using a standard primary amine couplingprotocol. PD-1 mAbs at 0.3 μg/ml were captured on anti-mouse Fc surfacefor 1 min at 10 μl/min PD-1/Fc in a serial dilutions from 3.3 nM to 120nM was injected over antibody-bound surface for 3 min at 30 μl/minfollowed by a 10 min dissociation phase. Association rates (K_(a) ork_(on)) and dissociation rates (K_(d) or k_(off)) were calculated usingthe one-to-one Langmuir binding model (BIA Evaluation Software, GE LifeSciences). The equilibrium dissociation constant (K_(D)) was calculatedas the ratio k_(off)/k_(on).

As shown in Table 1, both mu326 and mu517, a cognate sequence familymember related to mu317, have a sub-nanomolar K_(D) equaling to 0.324 nMand 0.289 nM, respectively, which is significantly better than that ofmu134. The K_(on) rate was similar among the three mAbs listed in Table1, yet the K_(off) rate was significantly different, much fasterdissociation rate was observed in mu134.

TABLE 1 Binding constant of certain top antibodies mAbs K_(on) (M⁻¹,s⁻¹) K_(off) (s) K_(D) (M) mu326 2.4 × 10⁵ 7.79 × 10⁻⁵ 3.24 × 10⁻¹⁰mu517 1.96 × 10⁵  5.66 × 10⁻⁵ 2.89 × 10⁻¹⁰ mu134 1.1 × 10⁵ 3.69 × 10⁻⁴3.35 × 10⁻⁹ 

Affinity Determination of Anti-PD-1 Fabs by SPR

Anti-PD-1 mAbs were converted into Fab version by PCR to fuse thevariable regions of heavy and light chains to the N-terminus of humanIgG2-CH1 and constant region of kappa chain, respectively, and subclonedin pcDNA3.1 vector (Invitrogen). Both expression vectors wereco-expressed in 293-F cells using a transient transfection protocolsimilar to the transient expression of whole antibodies. Briefly, theFab kappa chain was PCR amplified and subcloned in pcDNA3.1-basedexpression vector (Invitrogen, Carlsbad, Calif., USA). In a separateplasmid, the heavy chain variable region (VH) together with the CH1coding sequence from human IgG2 was fused with a C-terminal c-Myc-His8tag by overlapping PCR, and then subcloned in the expression vector. TheC232S and C233S (Kabat residue numbering, Kabat et al. Sequence ofproteins of immunologic interest, 5^(th) ed Bethesda, Md., NIH 1991)mutations were introduced in the IgG2 heavy chain to prevent disulfidebond exchange and stabilize human IgG2 in the IgG2-A conformation(Lightle et al. 2010 Protein Sci 19(4): 753-762). Both constructscontained a signal peptide upstream of the Fab mature sequences.Secreted expression of Fab was achieved by co-transfection of above 2plasmids into 293-F cells and cell culture supernatants were harvested6-7 days post transfection. His8-tagged Fabs were purified from cellculture supernatants using a Ni-sepharose Fast Flow column (Cat. No.17531801, GE Life Sciences) followed by size exclusion chromatographyusing a HiLoad 16/60 Superdex200 column (Cat. No. 17106901, GE LifeSciences). The purified Fabs were concentrated to 0.5-5 mg/mL in PBS andstored in aliquots in −80° C. freezer.

For affinity determinations of anti-PD-1 Fabs, SPR assays were used withthe BIAcore™ T-200 instrument (GE Life Sciences). Briefly, humanPD-1/His or cynomolgus monkey PD-1/His was coupled to activated CM5biosensor chips (Cat. No. BR100530, GE Life Sciences) to achieveapproximately 100-200 response units (RU), followed by blockingun-reacted groups with 1M ethanolamine Fab samples of increasingconcentration from 0.12 nM to 90 nM were injected in the SPR runningbuffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween20, pH7.4) at 30 μL/minute,and binding responses on human PD-1/His or monkey PD-1/His werecalculated by substracting of RU from a blank flow-cell. Associationrates (k_(on)) and dissociation rates (k_(off)) were calculated usingthe one-to-one Langmuir binding model (BIA Evaluation Software, GE LifeSciences). The equilibrium dissociation constant (K_(d)) was calculatedas the ratio k_(off)/k_(on).

The SPR-determined binding affinities of anti-PD-1 Fabs were listed inTable 18. Each anti-PD-1 Fab bound with high affinity (K_(d=0.15)-1 nM)to human PD-1. All Fabs, except 326-3G1, bound with slightly lower butcomparable (within 5 fold in K_(d)) affinities to cynomolgus monkeyPD-1.

Example 3 Functional Activity of Anti-PD-1 Antibodies in Human T Cells

Generation of Stable Cell Lines

Retroviral packaging cell line PT67, human T cell lines HuT78 and HEK293were obtained from the American Type Culture Collection (ATCC,Rockville, Md.). A HuT78 subline HuT78/PD-1 that expresses PD-1 wasgenerated by retroviral transduction using pFB-neo vector(Strategene/Agilent Tech, Santa Clara, Calif.) containing the PD-1 gene,according to the protocol described previously (Zhang et al. 2005 Blood106: 1544-1551). The T cell engager, a membrane-anchored chimeric Ab(OS8), was constructed by fusing the single chain variable fragment(scFv) of an anti-human CD3 mAb OKT3 (Kipriyanov et al. 1997, PEDS10:445-453) to the C-terminal domain (113-220) of mouse CD8α (NCBIAccession No: NP_(—)001074579.1) which includes hinge, transmembrane andcytoplasmic domains. By doing so, anti-CD3 scFv is anchored to cellsurface as a T cell activator. Human PD-L1, PD-L2 and OS8 cDNAs weresub-cloned into pcDNA3.1 vector. Stable cell lines HEK293/OS8/PD-L1,Hep3B/OS8/PD-L1 and HEK293/OS8/PD-L2 that co-express both OS8 and PD-L1or PD-L2 cDNAs were generated by co-transfection of HEK293 and Hep3Bcells (ATCC) with the paired plasmids, followed by hygromycin or G418selection for 10-14 days. Cell lines were then cloned by limitingdilution as described previously (Fuller S A, et al. Curr Protoc MolBiol. Chapter 11:Unit 11.8., 2001) Chimeric PD-1 receptor, named P3Z,was constructed by fusing the extracellular and transmembrane domains)of human PD-1 to the cytoplasmic domain of human CD3 ζ chain (NCBIAccession No. NP_(—)932170.1). P3Z-coding cDNA sequence was cloned intopFB-neo and delivered into HuT78 cells via retroviral transduction togenerate HuT78/P3Z cells.

Determination of PD-1 Antibody Functions by IL-2 Release in HuT78/PD-1Cells

To determine whether anti-PD-1 antibodies can block the interaction ofPD-L1-induced PD-1 signaling, HuT78/PD-1 cells (1.5×10⁴ cells per wellin 96-well plate) were pre-incubated with hybridoma supernatants or PD-1antibodies for 15 minutes prior to co-culture with HEK293/OS8/PD-L1 orHEK293/OS8/PD-L2 cells (4×10⁴ per well) in a flat bottom plate fed with200 μl of RPMI1640 growth medium per well at 37° C. After 16-18 hours,supernatants of the co-culture were collected. IL-2 was assayed by ELISAusing human IL-2 Ready-Set-Go! ELISA kits (Cat. No. 88-7025,eBiosciences, San Diego, Calif.). In this assay, blockade of PD-1signaling with anti-PD-1 antibodies resulted in enhanced TCR signalingand IL-2 production (FIG. 4).

As shown in FIG. 5 and Table 2, murine anti-PD-1 mAb, mu317 and mu326,elicited significantly higher functional activity than mu30, inhibitingPD-L1-induced PD-1 signaling which leads to increased IL-2 secretion.Both had higher IL-2 secretion (top line, Table 2), 675 and 634 pg/ml,respectively, and both had lower EC₅₀ (Effective concentration of mAb at50% level of IL-2 secretion induction) than mu30 antibody.

TABLE 2 IL-2 release induced by anti-PD-1 mAbs in HuT78/PD-1 cellsco-cultured with HEK293/OS8/PD-L1 cells Antibody Baseline (pg/ml) Topline (pg/ml) EC₅₀ (μg/ml) mu30 95 527 0.229 mu317 95 675 0.083 mu326 95634 0.053 mIgGs 95 N/A N/A Baseline: Average IL-2 release induced bymIgGs at all tested concentrations, see FIG. 4. Top line: Highest IL-2release based on regression calculation by Prizm Software, FIG. 4. N/A:Not applicable

Not only did the engagement of HuT78/PD-1 cells by anti-PD-1 mAbs blockPD-L1 induced T-cell activation, but also blocked PD-L2 induced IL-2release. Table 3 presented the data showing mu317 and mu326 had muchhigher potency in activating the T-cells as indicated by the parameters(EC₅₀) of IL-2 secretion than those of mu476.

TABLE 3 IL-2 release induced by anti-PD-1 mAbs in HuT78/PD-1 cellsco-cultured with HEK293/OS8/PD-L2 cells Antibody Baseline (pg/ml) Topline (pg/ml) EC₅₀ (μg/ml) 476 180 599 0.183 317 192 563 0.032 326 218635 0.038 Baseline: Average IL-2 release induced in the lower tail partof the sigmoid reaction curve. Top line: Average IL-2 release induced atthe plateau part of the sigmoid reaction curve

Determination of PD-1 Antibody Functions By Reverse Signaling of IL-2Release in HuT78/P3Z Cells

In chimeric receptor P3Z, PD-1 signaling domain was replaced with thecytoplasmic domain of CD3ζ. Therefore, P3Z mediates activation uponengagement with PD-L1, rather than inhibition as original PD-1 receptor.In this assay, HuT78/P3Z cells (3×10⁴/well) were pre-incubated withhybridoma supernatants or PD-1 antibodies for 15 minutes prior toco-culture with HEK293/PD-L1 or HEK293/PD-L2 cells (5×10⁴/well) in96-well flat bottom plates (a total volume of 200 μl/well) at 37° C.After 16-18 hours, supernatants were collected and IL-2 production wasassayed by ELISA as described above.

The functional activity of murine anti-PD-1 mAbs was further confirmedby direct read-out of T-cell activation in reverse signaling assaydescribed above. Consistent to the result described above, mu317 andmu326 had best functional activity among the mAbs we screened. As shownin Table 4 and Table 5, mu317 and mu326 were much more potent than oneof the low activity mAbs, mu37, both in terms of IC₅₀ and maximuminhibition.

TABLE 4 Inhibition of IL-2 secretion by anti-PD-1 mAbs in HuT78/P3Zcells co-cultured with HEK293/PD-L1 cells Antibody IC₅₀ (μg/ml) Maxinhibition, %  37 0.287 86.9 317 0.083 99.9 326 0.039 97.6 Maximuminhibition was calculated as percentage (%) of inhibition with anti-PD-1mAbs added to the highest level of 10 μg/ml in culture.

TABLE 5 Inhibition of IL-2 secretion by anti-PD-1 mAbs in HuT78/P3Zcells co-cultured with HEK293/PD-L2 cells Antibody IC₅₀ (μg/ml) Maxinhibition, %  37 0.127 43.3 317 0.020 94.3 326 0.018 93.4 Maximuminhibition was calculated as percentage (%) of inhibition with anti-PD-1mAbs added to the highest level of 10 μg/ml in culture.

Example 4 Activation of IFN-γ secretion by Anti-PD-1 mAb in PrimaryHuman PBMCs Co-Cultured with HEK293/OS8/PD-L1 Cells

To verify if the selected top mAbs against PD-1 also exert functionaleffect on primary human immune cells, we assayed the antibody functionby using freshly isolated peripheral blood mononuclear cells (PBMCs),which are mainly consisted of T-cells (50-70%), B-cells and NK cells(15-30%), and monocytes (2-10%). Human PBMCs were isolated from healthydonors by density gradient centrifugation using ficoll lymphocyteseparation medium (Histopaque-1077; Sigma-Aldrich, MO) according to themanufacturer's instructions. All the human blood collection followed theInternal Procedure of Beigene. PBMCs were then stimulated with anti-CD3mAb (40 ng/mL) OKT3 (Cat. No. 16-0037, eBioscience, CA) for 3 days priorto assay. FACS analysis (Example 1) showed that PD-1 expression on theactivated PBMCs (primarily T cells) was increased to variable degreedependent on individual donors (Table 6). To determine the response ofpre-activated T cells to PD-1 ligand-positive tumor cells uponengagement TCR/CD3 complex, PBMCs (1×10⁴) were co-cultured with eitherHEK293/OS8/PD-L1 or HEK293/OS8/PD-L2 cells (3×10⁴) in 96-wellflat-bottom plates for 15-18 hours. Cell-free supernatants were assayedfor IFN-γ level by ELISA using Ready-Set-Go! ELISA kits (Cat. No.88-7316, eBiosciences), which is the most prominent indicator of T-cellactivation, as well as of other immune cell activation (Thakur A. et al.2012 Vaccine, 30:4907-4920).

Percent gated PD-1 staining positive cells versus total PMBCs stainedPBMCs and treatment Donor-3 Donor-4 PBMCs, not stimulated/ 12.0% 3.2%stained by PD-1 Ab PBMCs, stimulated/ 40.0% 38.1% stained by PD-1 AbPBMCs, not stimulated/ ≦0.5% ≦0.5% stained by control Ab PBMCs,stimulated/ ≦0.5% ≦0.5% stained by control Ab Stimulation: freshlyisolated PBMCs were cultured for 3 days in presence of anti-CD3antibody, OKT3, and IL-2. Without stimulation: fresh PBMCs subjected toantibody staining and FACS analysis.

FIG. 6 demonstrated that presence of mAbs mu317 and mu326 in theco-culture of pre-activated PBMCs and HEK293/OS8/PD-L1 cells resulted inincreasing IFN-γ accumulation in a dose-dependent manner. Although thebase level of IFN-γ with control murine IgG treatment varies amongdifferent donors, the increase of IFN-γ secretion in PBMCs treated bymu317 or mu326 is statistically significant in the range of 0.1 to 10μg/ml of antibody treatment. Comparing to the corresponding level ofmIgG-treated PBMCs, IFN-γ secretion induced by mu317 and mu326 betweenthe 0.1 to 10 μg/ml concentration levels increased 2.5 to 3.2 fold inPBMCs from Donor-19, and increased 1.4 to 2.3 fold in PBMCs of Donor-20,respectively.

Example 5 Activation of Human NK Cells by Anti-PD1 mAbs

Stable Cell Lines for Functional Assay in NK Cells

Primary human NK cells were reported previously to express PD-1 proteinin response to IL-2 treatment and inhibiting PD-1-mediated signalingenhanced cytotoxicity of NK cells (2010 Blood, 116: 2286). Forquantitative assay of functional effect exerted by anti-PD-1 mAbs in NKcells, human NK cell line NK92MI (ATCC) and lung cancer cell lineSK-Mes-1 (ATCC) were engineered to stably express human PD-1 and PD-L1,respectively, by retroviral transduction according to the protocolsdescribed previously (Zhang et al. 2005, Blood 106: 1544-1551, Zhang etal. 2006, Cancer Res, 66: 5927). The two stable cell lines were named asNK92MI/PD-1 and SK-Mes-1/PD-L1

Anti-PD-1 Abs Promote IFN-γ Production and Secretion in NK92MI/PD-1Cells

Functional activity of the anti-PD-1 mAbs on NK cells was assayed byquantitative measurement of IFN-γ production and secretion inNK92MI/PD-1 cells which were co-cultured with lung cancer cell lineSK-MES-1/PD-L1 at ratio of 1 to 2 in 96-well flat-bottom plate withtotal of 6×10⁴ cells per well. The anti-PD-1 mAbs were added toNK92MI/PD-1 cells 15 minutes before the co-culture started, then thecells were co-cultured for overnight in CO₂ incubator. Cell-freesupernatants were assayed for ITN-γ level by ELISA as described inExample 4.

All anti-PD-1 mAbs trigged significant increase of IFN-γ production fromthe baseline with low concentration of antibody treatment to top linewith high concentration of antibody treatment. The two top antibodies,mu317 and mu326, had lower EC₅₀, than the comparison antibody 5C,indicating they have more potent activating effect to the NK cells(Table 7).

TABLE 7 IFN-γ secreted in medium (pg/ml) by NK92MI/PD-1 cells inpresence of anti-PD-1 mAb and SK-MES-1/PD-L1 cells Antibody Baseline(pg/ml) Top line (pg/ml) EC₅₀ (μg/ml) 317 28 532 0.40 326 15 509 0.20 5C20 535 1.17 Baseline: Average IFN-γ release induced in the lower tailpart of the sigmoid reaction curve. Top line: Average IFN-γ releaseinduced at the plateau part of the sigmoid reaction curve

Anti-PD-1 Antibody Enhances Cancer Cell Killing Mediated by NK92MI/PD-1Cells

Cytotoxicity of NK92MI/PD-1 cells against SK-MES-1/PD-L1 cells wasdetermined by lactate dehydrogenase (LDH) release assay using theCytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison,Wis.). In brief, NK92MI/PD-1 cells (10⁵) were preincubated withanti-PD-1 mAbs at final concentrations within the range of 0.004-10μg/ml for 15 minutes, and SK-MES-1/PD-L1 cells (2×10⁴) were added to theimmune cell culture in a 96-well V-bottom plate at an effector to tumorcell (E:T) ratio of 5:1, then co-cultured for 5 hours. The completetumor cell lysis was set as maximum cell killing, the LDH-release assayreadout of each sample was calculated as percentage of maximum cellkilling. The cell killings (%) of all samples were normalized cross theplates using 10% of baseline as the common standard.

In the specific cytotoxicity assay set as above, the selected anti-PD-1mAbs caused a net tumor cell killing (=top line−baseline) ranging from19% to 20.2% at high concentration of mAb input. Mu317 and mu326 hadlower EC50 than mu336, indicating better potency to trigger NK92MI/PD-1cell-mediated tumor cell killing (Table 8).

TABLE 8 Cytotoxicity of NK92MI/PD-1 cells towards tumor cells induced byanti-PD-1 mAb Antibody Baseline (%) Top line (%) EC₅₀ (μg/ml) 317 1029.06 0.50 326 10 30.19 0.37 336 10 29.72 1.52 Baseline: Percent oftumor cells killed not due to the effect of anti-PD-1 mAbs, normalizedto 10% cross plates. Top line: Average percent of tumor killed inpresence of highest concentrations of mAbs, i.e. 3 μg/ml and 10 μg/ml

Example 6 Cloning and Sequence Analyses of PD-1 mAbs

The murine hybridoma clones secreting a specific mAb were cultured to adensity of 3 to 10×10⁶ cells in a 100 mm-tissue culture dish, and thecells were harvested through centrifugation at 1500 rpm in a swingbucket rotor. Total cellular RNA was isolated using Ultrapure RNA kit(Cat. No. CW0581, CWBIOTECH, Beijing, China) following themanufacturer's protocol. The RNA was resuspended in double-deionizedwater, concentration measured by NanoDrop (ThermoFisher, Shanghai,China).

PCR primers used for mAb cDNA cloning were synthesized by Invitrogen(Beijing, China) based on the sequences reported previously (Brocks etal. 2001 Mol Med 7:461-469). The 1^(st) strand cDNA was synthesizedusing reverse transcriptase (Cat. No. AH301-02, Transgen Biotech,Beijing, China). PCR amplification of specific mAb cDNA was performedusing PCR reagent kit (Cat. No. Ap221-12, TransGen Biotech, Beijing,China) and following manufacturer's protocol. The PCR product was eitherdirectly sequenced by service provider (GeneWiz, Beijing, China) orsubcloned into a pCR vector (Invitrogen), subsequently sequenced(GeneWiz).

The protein sequences of murine mAbs were analyzed by sequence homologyalignment. MAbs were grouped based on sequence homology andepitope-mapping results (Example 13). Complement determinant regions(CDRs) were identified based on Kabat (Wu, T. T. and Kabat, E. A., 1970J. Exp. Med. 132: 211-250) and IMGT system (Lefranc M.-P. et al., 1999Nucleic Acids Research, 27, 209-212) by sequence annotation and byinternet-based sequence analysis(http://www.imgt.org/IMGT_vquest/share/textes/index.html andhttp://www.ncbi.nlm.nih.gov/igblast/). As shown in Table 9, the CDRs ofmu317 and mu326 are very different in sequence length and identity.

TABLE 9 CDRs of mu317 and mu326 SEQ SEQ SEQ ID ID ID MAbs CDR1 NO CDR2NO CDR3 NO mu317, HC GFSLT 

11 V IWAGGST NYNSALMS 12 ARAYGNYWYIDV 13 mu317, LC  KAS QSVSND VA 14 YAFHRFT 15 HQAYSSPYT 16 mu326, HC GYTFTNYG MN 17 W INNNNGEP TYAEEFKG  18ARDVMDY 19 mu326, LC RAS ESVDNYGYSF MH 20 RAS NLES 21 QQSKEYPT 22 Note:CDRs in bold face are based on Kabat system; CDRs underlined are basedIMGT system.

Example 7 Humanization of the Murine mAbs

Simulation of Antibody 3D Structure

The three dimensional structures were simulated for variable domains ofmu317 and mu326 in order to identify framework residues that might beimportant for supporting CDR loop structures. Potentially importantframework residues were kept as the original murine residues in thefirst round antibody humanization. The previously established structuralmodeling method for antibodies (Morea et al. Methods 2000 20:267-279)was adopted to simulate 3D structure of anti-PD-1 mAbs based on theknown canonical structures of antibodies (Al-Lazikani et al. 1997Journal of Molecular Biology 273:927-948). Briefly, the sequence of eachvariable domain (Vk and Vh) of murine antibody was blasted in the PDBdatabase (Protein Data Bank, http://blast.ncbi.nlm.nih.gov/) to identifythe most homologous antibody sequence with known high resolutionstructure (resolution less than 2.5 angstrom). Selected structuretemplates for modeling mu317 and mu326 (listed in Table 10) had the sameclasses of canonical loop structures in L-CDR1, L-CDR2, L-CDR3, H-CDR1,and H-CDR2 to the target antibodies to be modeled. If the templates forthe Vk and the Vh came from different immunoglobulins, they were packedtogether by a least-squares fit of the main chain atoms to form a hybridstructure of Vk-Vh interface residues, which was used as the templatesfor structural homology modeling by Swiss-model program (Kiefer et al.2009 Nucleic Acids Research 37, D387-D392). Certain side chainconformation was adjusted while the main chain conformations wereretained. At the sites where the parent structure and the modeledstructure had the same residue, the side chain conformation wasretained. At sites where the residues were different, side chainconformations were modeled on the basis of template structure, rotamerlibraries and packing considerations. After homology modeling, PLOPprogram (Jacobson et al. 2002 Journal of Physical Chemistry106:11673-11680) was used to refine the homology models to minimizeall-atom energy and optimize Vk and Vh interface. This step wasperformed to improve the stereochemistry, especially in those regionswhere segments of structures coming from different antibodies had beenjoined together.

TABLE 10 Structure templates used in antibody structure simulations PDBcode of template structure Sequence Sequence Antibody chain (PDBtemplate for H-CDR3) identity similarity mu317 Vk 3MXV 87% 92% mu317 Vh3VFG 83% 91% mu326 Vk 1EJO 92% 94% mu326 Vh 1NCA 88% 90% 317-1 Vk 4HJJ90% 95% 317-1 Vh 3VFG (1AY1) 75% 87% 326-1 Vk 1EJO 87% 92% 326-1 Vh 3T2N(3CXD) 84% 86%

The structures were also simulated for CDR-grafted 317-1 and 326-1 inorder to guide further rounds of antibody engineering to enhance theextents of humanization and/or enhance antibody stabilities. Theselected structure templates are also listed in Table 10. The structuresimulations were done in a similar way to above procedure, except thatthe possible conformations of H-CDR3 were taken from PDB templates 1AY1for 317-1 and 3CXD for 326-1, respectively, which contained H-CDR3s ofsimilar size and torso region. Energy minimization for grafted H-CDR3residues was done using PLOP.

Humanization

For humanization of the anti-PD-1 mAbs, we searched human germline IgGgenes homologous to the cDNA sequences of mu317 and mu326 variableregions by blasting the human immunoglobulin gene database in IMGT(http://www.imgt.org/IMGT_vquest/share/textes/index.html) and NCBI(http://www.ncbi.nlm.nih.gov/igblast/) websites. The human IGVH and IGVκwith high homology to the PD-1 mAbs were selected as the template forhumanization.

Humanization was carried out in principle by CDR-grafting. In the 1^(st)round of humanization, mutations from murine to human amino acidresidues in framework sequences of variable regions was guided by thesimulated 3D structures, and only the murine amino acid residues whosechanges retain the overall antibody and CDR loop structure were mutatedto human sequence as described above. The initial versions of humanizedmAbs were hu317-1 (SEQ NO 47-50) and hu326-1 (SEQ NO 55-58), whichcomprise a heavy chain with humanized variable heavy chain (Vh) fused tohuman IgG2 constant region (NCBI accession No. P01859) and a light chainwith humanized variable light chain kappa (Vκ) fused to human Ig kappaC-region (NCBI Accession No. P01834). Likewise, we generated chimericantibodies from mu317 and mu326, which are consisted of a murine VHfused to human IgG2 constant region and a murine Vκ fused to human Igkappa C-region. The full chimeric antibodies were named as ch317 andch326, respectively. All recombinant mAbs were expressed and purified asdescribed in Example 1.

FACS and functional assays demonstrated that mAb hu317-1 almost retainedthe same binding and functional activity as the mu317 and ch317. TheEC₅₀ difference in FACS analysis between mu317 versus ch317 and hu317-1may be interpreted by the fact that two different detection antibodies,a goat anti-mouse IgG and a goat anti-human IgG, were used in FACS. Inthe two functional assays, all three versions of 317 were treated moreequal, and the results also close to each other (Table 11).

As result of the initial round of humanization for mu326, mAb hu326-1retained similar functional feature to the parental ch326 and mu326although functional activity in FACS binding assay and in HuT78/PD-1cell-based IL-2 release assay may be slightly weaker than ch326 (Table12).

TABLE 11 Comparison of mu317, ch317 and hu317-1 by FACS and functionalassays Parameter Assay mu317 ch317 hu317-1 FACS EC₅₀ (μg/ml) 0.11 0.360.46 Top MFI* 205 217 203 Assay-1 EC₅₀ (μg/ml) 0.11 0.08 0.09 Top line(pg/ml) 346 294 386 Baseline (pg/ml) 98 82 91 Assay-2 IC₅₀ (μg/ml) 0.110.10 0.11 Max inhibition 99.5% 99.0% 99.8% *MFI: mean fluorescenceintensity from FACS analysis Assay-1: IL-2 release induced by the mAbsin HuT78/PD-1 cells co-cultured with HEK293/OS8/PD-L1 cells Assay-2:IL-2 release induced by the mAbs in HuT78/P3Z cells co-cultured withHEK293/PD-L1 cells

TABLE 12 Comparison of mu317, ch317 and hu317-1 by FACS and functionalassays Parameter Assay mu326 ch326 hu326-1 FACS EC₅₀ (μg/ml) 0.126 0.0720.117 Top MFI 195 163 129 Assay-1 EC₅₀ (μg/ml) 0.038 0.074 0.112 Topline (pg/ml) 1149 1057 1143 Baseline (pg/ml) 242 250 283 Assay-2 IC₅₀(μg/ml) 0.14 0.12 0.10 Max inhibition 96.9% 81.0% 84.4% Assay-1: IL-2release induced by the mAbs in HuT78/PD-1 cells co-cultured withHEK293/OS8/PD-L1 cells Assay-2: IL-2 release induced by the mAbs inHuT78/P3Z cells co-cultured with HEK293/PD-L1 cells

Based on the 1^(st) round of humanization, we further mutated the othermurine amino acid (AA) residues in the framework (FR) of hu317-1_Vh and_Vκ individually to assess the impact on the antibody function. As shownin Table 13, the seven individual mutations in Vh and one mutation in Vκof hu317-1 all have similar functional activities. Only minor changeswere observed in some Vh mutation, such as hu317-2_K71V with slightlyweaker inhibitory function among the mutations. However, when all themurine amino acid residues mutated together to human (hu317-3A), thefunction is clearly weaker than the rest mutations in FACS and IL-2release assays.

In the initial trial described above, hu326-1 reached significanthumanization level in the FR except for a few of murine AA residuesleft. Yet, it has weaker function than the mu326. Therefore, we mademore individual mutations either back to murine residues or forward tohuman residues to explore the contribution of each individual AA tomAb326 function. Table 14 presented all single AA mutations made basedon hu326-1_Vh template (SEQ NO 56, SEQ NO 57) and their functional assayresults. Majority of the mutations showed better functional activitythan those of hu326-1, matching the original mu326 mAb. A couple ofmutations (E46K and F95Y) showed slightly less potency in the EC₅₀ orIC₅₀, indicating the role of those residues in the antibody structureand function.

TABLE 13 Comparison of functional activity of Fabs with humanizationmutations in hu317-1 framework Fab and composition FACS, IL-2 release inHuT78/P3Z Vh Vκ EC₅₀ Max inhibition, % EC₅₀ hu317-1_Vh hu317-1_Vκ 0.1998.78 0.30 hu317-2_L48I hu317-1_Vκ 0.14 98.51 0.37 hu317-2_L67Vhu317-1_Vκ 0.15 98.57 0.30 hu317-2_K71V hu317-1_Vκ 0.18 96.55 0.48hu317-2_N73T hu317-1_Vκ 0.15 98.29 0.31 hu317-2_S76N hu317-1_Vκ 0.1398.56 0.28 hu317-2_V78F hu317-1_Vκ 0.18 98.03 0.38 hu317-2_M82Lhu317-1_Vκ 0.13 98.47 0.27 hu317-1_Vh HU317- 0.21 98.86 0.27 2_G100Qhu317-3A hu317-1_Vκ 0.32 79.66 0.35 Note: Unit for EC₅₀ is μg/ml;mutated amino acid residue numbering is same as in the listed sequencesfor hu317-1; hu317-3A has all the framework sequence mutated to human.

TABLE 14 Comparison of functional activity of mAbs with mutations inhu326-1 framework IL-2 release IL-2 release FACS, in HuT78/P3Z inHuT78/PD-1 EC₅₀ Max inhibition, IC₅₀, Top line, EC₅₀, Antibody μg/ml %μg/ml pg/ml μg/ml ch326 0.118 93.05 0.074 993 0.135 hu326-1 0.317 92.380.087 987 0.213 hu326-2 S9P^(B) 0.145 96.04 0.075 1022 0.136 hu326-2A16E^(B) 0.155 96.33 0.078 1048 0.126 hu326-2 E46K^(B) 0.132 95.25 0.0791244 0.259 hu326-2 G63D^(B) 0.139 96.44 0.064 1069 0.120 hu326-2A76V^(F) 0.102 96.65 0.071 1002 0.112 hu326-2 S84N^(B) 0.131 96.52 0.0601015 0.126 hu326-2 S85N^(B) 0.110 95.62 0.093 932 0.104 hu326-2 T88N^(B)0.098 95.85 0.102 hu326-2 F95Y^(F) 0.097 95.62 0.166 1028 0.135 ^(B)Backmutation to murine amino acid; ^(F)Forward mutation to human amino acid.All of the mutations were made in hu326-1_Vh (SEQ NO 56), which werepaired with hu326-1_Vk (SEQ NO 58).

To explore the best possible Vh and Vκ sequence composition for mAbs 317and 326 that could be used as therapeutics in human, we made a varietyof combination mutations (including some mutations in the CDR sequences)in considerations of the antibody features, such as humanization levelin FR, functional activities, physicochemical properties,antibody-dependent cell-mediated cytotoxicy (ADCC) andcomplement-dependent cytotoxicity (CDC). Most of the mutations weredeemed not passing the qualification standards. Through the engineeringprocess, six of the humanized, recombinant mAbs were selected for theirpotential therapeutic utility: hu317-4B2 (SEQ ID NO 43-44), hu317-4B5(SEQ ID NO 45-46), hu317-4B6 (SEQ ID NO 23-26), hu326-3B1 (SEQ ID NO51-52), hu326-3G1 (SEQ ID NO 53-54) and hu326-4A3 (SEQ ID NO 27-30). TheCDRs of the mAb were compared to those of original murine antibodies,shown in Table 15 and Table 16.

Among the six mAbs, hu317-4B2, hu317-4B5 and hu317-4B6 are closelyrelated to each other in sequences and very similar in their functionalactivities and strength. On the other hand, hu326-3B1, hu326-3G1 andhu326-4A3 are quite close to each other in sequences and functionalities(Table 17-18). Within each of the two groups of mAbs, they also sharedmany other features in addition to sequences and function, such asphysicochemical properties and binding epitopes (described in Examples10 and 11) though some minor differences do exist.

TABLE 15 Comparison of CDRs among different versions of mAbs 317 SEQ SEQSEQ mAbs CDR1 ID NO CDR2 ID NO CDR3 ID NO mu317, HC GFSLTSYGVH 11VIWAGGSTNYNSALMS 12 ARAYGNYWYIDV 13 hu317-1, HC GFSLTSYGVH 11VIWAGGSTNYNPSLKS 59 ARAYGNYWYIDV 13 hu317-4B2, HC GFSLTSYGVH 11VIYAGGSTNYNPSLKS 60 ARAYGNYWYIDV 13 hu317-4B5, HC GFSLTSYGVH 11VIYAGGSTNYNPSLKS 60 ARAYGNYWYIDV 13 hu317-4B6, HC GFSLTSYGVH 11VIYADGSTNYNPSLKS 32 ARAYGNYWYIDV 13 mu317, LC KASQSVSNDVA 14 YAFHRFT 15HQAYSSPYT 16 hu317-1, LC KASQSVSNDVA 14 YAFHRFT 15 HQAYSSPYT 16hu317-4B2, LC KSSESVSNDVA 61 YAFHRFT 15 HQAYSSPYT 16 hu317-4B5, LCKSSESVSNDVA 61 YAFHRFT 15 HQAYSSPYT 16 hu317-4B6, LC KSSESVSNDVA 61YAFHRFT 15 HQAYSSPYT 16 Note: AA residues underlined are changed frommurine sequence to human antibody sequences or for improvement ofphysicochemical properties.

TABLE 16 Comparison of CDRs among different versions of mAbs 326 SEQ SEQSEQ mAbs CDR1 ID NO CDR2 ID NO CDR3 ID NO mu326, HC GYTFTNYGMN 17WINNNNGEPTYAEEFKG 18 ARDVMDY 19 hu326-1, HC GYTFTNYGMN 17WINNNNGEPTYAQGFRG 62 ARDVMDY 19 hu326-3B1, HC GYTFTNYGMN 17WINNNNGEPTYAQDFRG 63 ARDVMDY 19 hu326-3G1, HC GYTFTNYGMN 17WINNNNGEPTYAQDFRG 63 ARDVMDY 19 hu326-4A3, HC GYTFTNYGMN 17WINNNNAEPTYAQDFRG 38 ARDVMDY 19 mu326, LC RASESVDNYGYSFMH 20 RASNLES 21QQSKEYPI 22 hu326-1, LC RASESVDNYGYSFMH 20 RASNLES 21 QQSKEYPI 22hu326-3B1, LC RASESVDNYGYSFMH 20 RASNLES 21 QQSKEYPI 22 hu326-3G1, LCRASESVDNYGYSFMH 20 RASNLES 21 QQSKEYPI 22 hu326-4A3, LC RASESVDNYGYSFMH20 RASNLES 21 QQSKEYPI 22 Note: AA residues underlined are changed frommurine sequence to human antibody sequences or for improvement ofphysicochemical properties.

TABLE 17 Binding activities of humanized mAbs assayed by ELISA and FACSmAbs ELISA, EC₅₀ μg/ml FACS, EC₅₀ μg/ml hu317-4B2 0.066 0.129* hu317-4B50.057 0.115* hu317-4B6 0.061 0.092* hu326-3B1 0.092 0.165 hu326-3G10.088 0.190 hu326-4A3 0.091* 0.142* *FACS data by using Fab version ofantibodies without normalization. ** Data from bridging study andnormalized.

TABLE 18 Binding affinity of Fabs assayed by SPR Fab K_(on) (M⁻¹, s⁻¹)K_(off) (s) K_(D) (M) hu317-4B5 3.89 × 10⁵ 9.07 × 10⁻⁵ 2.33 × 10⁻¹⁰hu317-4B6 5.71 × 10⁵ 8.37 × 10⁻⁵ 1.47 × 10⁻¹⁰ hu326-3B1 2.18 × 10⁵ 1.90× 10⁻⁴ 8.70 × 10⁻¹⁰ hu326-3G1 2.00 × 10⁵ 2.01 × 10⁻⁴ 1.00 × 10⁻⁹ 

Affinity Determination of Humanized Anti-PD-1 Fabs by SPR

Anti-PD-1 mAbs were converted into Fab version by PCR to fuse thevariable regions of heavy and light chains to the N-terminus of humanIgG2-CH1 and constant region of kappa chain, respectively, and subclonedin pcDNA3.1 vector (Invitrogen). Both expression vectors wereco-expressed in 293-F cells using a transient transfection protocolsimilar to the transient expression of whole antibodies. Briefly, theFab kappa chain was PCR amplified and subcloned in pcDNA3.1-basedexpression vector (Invitrogen, Carlsbad, Calif., USA). In a separateplasmid, the heavy chain variable region (VH) together with the CH1coding sequence from human IgG2 was fused with a C-terminal c-Myc-His8tag by overlapping PCR, and then subcloned in the expression vector. TheC232S and C233S (Kabat residue numbering, Kabat et al. Sequence ofproteins of immunologic interest, 5^(th) ed Bethesda, Md., NIH 1991)mutations were introduced in the IgG2 heavy chain to prevent disulfidebond exchange and stabilize human IgG2 in the IgG2-A conformation(Lightle et al. 2010 Protein Sci 19(4): 753-762). Both constructscontained a signal peptide upstream of the Fab mature sequences.Secreted expression of Fab was achieved by co-transfection of above 2plasmids into 293-F cells and cell culture supernatants were harvested6-7 days post transfection. His8-tagged Fabs were purified from cellculture supernatants using a Ni-sepharose Fast Flow column (Cat. No.17531801, GE Life Sciences) followed by size exclusion chromatographyusing a HiLoad 16/60 Superdex200 column (Cat. No. 17106901, GE LifeSciences). The purified Fabs were concentrated to 0.5-5 mg/mL in PBS andstored in aliquots in −80° C. freezer.

For affinity determinations of anti-PD-1 Fabs, SPR assays were used withthe BIAcore™ T-200 instrument (GE Life Sciences). Briefly, humanPD-1/His or cynomolgus monkey PD-1/His was coupled to activated CM5biosensor chips (Cat. No. BR100530, GE Life Sciences) to achieveapproximately 100-200 response units (RU), followed by blockingun-reacted groups with 1M ethanolamine. Fab samples of increasingconcentration from 0.12 nM to 90 nM were injected in the SPR runningbuffer (10 mM HEPES, 150 mM NaCl, 0.05% Tween20, pH7.4) at 30 μL/minute,and binding responses on human PD-1/His or monkey PD-1/His werecalculated by substracting of RU from a blank flow-cell. Associationrates (k_(on)) and dissociation rates (k_(off)) were calculated usingthe one-to-one Langmuir binding model (BIA Evaluation Software, GE LifeSciences). The equilibrium dissociation constant (K_(d)) was calculatedas the ratio k_(off)/k_(on).

The SPR-determined binding affinities of anti-PD-1 Fabs were listed inTable 18. Each anti-PD-1 Fab bound with high affinity (K_(d)=0.15-1 nM)to human PD-1. All Fabs, except 326-3G1, bound with slightly lower butcomparable (within 5 fold in Kd) affinities to cynomolgus monkey PD-1.

Example 8 Generation and Expression of Ecombinant Anti-PD-1 mAbs withModified Human IgG4 Constant Region

Since PD-1 is primarily expressed in activated T cells, PD-1 blockingantibodies linked to naturally occurring type of IgG-Fe moieties areexpected to induce Fc-mediated effector functions, such as ADCC and CDC,to a variable degree depending on the IgG subclasses, which results inelimination of activated T cells (Natsume A, et al, 2009 Drug Des DevelTher. 3: 7-16). Human antibody subclass IgG4 was shown in many previousreports that it has modest ADCC and almost no CDC effector function(Moore G L, et al. 2010 MAbs, 2:181-189). On the other hand, naturalIgG4 was found less stable in stress conditions such as in acidic bufferor under increasing temperature (Angal, S. 1993 Mol Immunol, 30:105-108;Dall'Acqua, W. et al, 1998 Biochemistry, 37:9266-9273; Aalberse et al.2002 Immunol, 105:9-19). In order to spare PD-1⁺T cells from beingkilled and to improve physicochemical properties of the anti-PD-1antibodies, the humanized mAbs were linked to IgG4 engineered bycombinations of mutations to have reduced or null FcγR binding or C1qbinding activities, therefore, attenuating or eliminating ADCC and CDCeffector functions. Considering physicochemical properties of antibodyas a biological drug, one of the less desirable, intrinsic properties ofIgG4 is dynamic separation of its two heavy chains in solution to formhalf antibody, which lead to bi-specific antibodies generated in vivovia a process called “Fab arm exchange” (Van der Neut Kolfschoten M, etal. 2007 Science, 317:1554-157). The mutation of serine to proline atposition 228 (EU numbering system) appeared inhibitory to the IgG4 heavychain separation (Angal, S. 1993 Mol Immunol, 30:105-108; Aalberse etal. 2002 Immunol, 105:9-19). Some of the amino acid residues in thehinge and γFc region were reported to have impact on antibodyinteraction with Fcγ receptors (Chappel S M, et al. 1991 Proc. Natl.Acad. Sci. USA, 88:9036-9040; Mukherjee, J. et al., 1995 FASEB J,9:115-119; Armour, K. L. et al., 1999 Eur J Immunol, 29:2613-2624;Clynes, R. A. et al, 2000 Nature Medicine, 6:443-446; Arnold J. N., 2007Annu Rev Immunol, 25:21-50). Furthermore, some rarely occurring IgG4isoforms in human population may also elicit different physicochemicalproperties (Brusco, A. et al. 1998 Eur J Immunogenet, 25:349-55;Aalberse et al. 2002 Inununol, 105:9-19). However, lumping all themutations and isoforms previously discovered into a specific antibodydoes not warrant for an ideal antibody molecule to share all thefeatures for therapeutics such as described above, which may be resultedfrom contradictory effect of the combined mutations and from impact ofvariable region to the effector function and physicochemical propertiesof an antibody (Igawa T. et al., 2010 Prot Eng Design Select,23:385-392; Perchiacca J. M. and Tessier P. M., 2012 Ann Rev Biomol Eng3:263-286).

To generate anti-PD-1 mAbs with least ADCC, CDC and instability, wemodified the hinge and γFc region of human IgG4 by introduce a number ofcombinations of mutations, which created IgG4mt1 to IgG4mt12. Some ofthe modified IgG4 variants were clearly less desirable as indicated byour assay results, several relevant IgG4 variants and modified sequenceswere listed in Table 19. The assessment of these antibodies is describedherein.

TABLE 19 Sequence modifications of IgG4 variants IgG4 and Amino acidresidues* variants . . . 228 229 230 231 232 233 234 235 236 . . . 265 .. . 309 . . . 409 . . . IgG4 . . . S C P A P E F L G . . . D . . . L . .. R . . . IgG4mt1 . . . P C P A P E F L G . . . D . . . L . . . R . . .IgG4mt2 . . . P C P A P P V A G . . . D . . . L . . . R . . . IgG4mt6 .. . P C P A P P V A G . . . A . . . L . . . R . . . IgG4mt8 . . . P C PA P P V A G . . . T . . . L . . . R . . . IgG4mt9 . . . P C P A P P V AG . . . A . . . L . . . K . . . IgG4mt10 . . . P C P A P P V A G . . . A. . . V . . . K . . . *Amino acid numbering is based on EU system.Changes are highlighted by underline.

Example 9 IgG4mt10 has no FcγR Binding, Lowest ADCC and CDC EffectorFunction

ADCC is initiated when an antibody binds to cell surface target proteinfollowed by ligation to Fcγ receptors (FcγRs) expressed on effectorcells. It was well documented that human IgG1 has significantly higherbinding affinity to FcγRs than IgG2 and IgG4, specially, binding toFcγR-I and FcγR-IIIA, which correlated to the strength of IgG1 toactivate ADCC. Reminiscent of ADCC, CDC is activated when an antibodycross-links a cell surface target and C1q protein, which followed by acascade reaction of complement complex formation and target cell lysis.As proxy of ADCC and CDC, assays for antibody binding to FcγRs and C1qmay serve as the fundamental indicator of ADCC and CDC. We thereforesystematically assessed the mAbs binding to all the major FcγRs.

FcγR Binding

Binding of various IgG4 mutants to FcγRs was determined by flowcytometry. In brief, a series of HEK293 transfectants expressing humanFcγRs were established. These transfectants expressed FcγRI, FcγRIIA,FcγRIIB or FcγRIIIA. Multi-subunit FcγRs (i.e., FcγRI and FcγRIIIA) wereco-expressed with FcRγ. Polymorphic variants (i.e., FcγRIIA H131 andR131, FcγRIIIA F158 and V158) were also included. A secondary antibody(goat anti-human IgG F(ab)'2-Alexa Fluor 488, Jackson ImmunoResearch,West Grove, Pa., USA) was used to detect the binding of anti-PD-1 mAbswith modified IgG4 variants (Table 19) to FcγR⁺ HEK293 cells. Asexpected, anti-PD-1 mAbs in IgG1 format (hu317-1/IgG1 andhu317-4B6/IgG1) bind strongly to all FcγRs including FcγRI, FcγRIIA(H131 and R131 alleles), FcγRIIB, and FcγRIIIA (V 158 and F158 alleles)(Table 20). Interestingly, when the two different version ofhumanization mAbs, hu317-1 and hu317-4B6 (with differences in both Vhand Vκ), were generated in the same IgG4 variant format, such as eitherin IgG4mt1 or in IgG4mt6 format, their binding strength (MFI) vary by anrange from a couple fold to close to 100 fold (e.g. 455.2/115.7=3.9fold; 13.6/1.0=13.6 fold; 434.6/4.9=88.7 fold; and etc., see Table 20).It is consistent to the previous findings by other that the variableregions of antibodies do have significant impact on the binding to FcRs,therefore, exerting the impact on effector function such as ADCC (IgawaT. et al., 2010 Prot Eng Design Select, 23:385-392; Perchiacca J. M. andTessier P. M., 2012 Ann Rev Biomol Eng 3:263-286).

As demonstrated in Table 20, when hu317-4B6 and hu326-4A3 were made inIgG4mt 10 format, they have the lowest binding activity to FcγRs amongthe PD-1 mAbs and IgG variant formats listed in the table, as well asmany other humanization mAbs and IgG formats we have tested in thestudy. The uniqueness of hu317-4B6 and hu326-4A3 in IgG4mt10 format inthis regard may not be extended to the same family of humanization mAbswith somewhat distant sequence homology, such as hu317-1, as describedabove.

TABLE 20 Binding strength (MFI*) of anti-PD-1 mAbs to Fc × Rs determinedby FACS FcγRIIA FcγRIIA FcγRIIIA FcγRIIIA mAbs FcγRI (H131) (R131)FcγRIIB (F158) (V158) hu317-1/ 2152.9 168.7 139.6 442.4 99.7 277.2 IgG1hu317-4B6/ 2771.7 1.7 0.6 1.9 28.0 293.7 IgG1 hu317-I/ 455.2 21.3 21.9434.6 0.6 20.7 gG4mt1 hu317-4B6/ 115.7 0.2 0.0 4.9 0 6.1 IgG4mt1hu317-1/ 13.6 1.0 0.8 1.8 0.9 1.1 IgG4mt6 hu317-4B6/ 1.0 0 0 0 0 0IgG4mt6 hu317-4B6/ 0.4 0 0 0 0 0 IgG4mt10 hu326-4A3/ 0.5 0 0 0 0 0IgG4mt10 *MFI: mean fluorescence intensity from FACS analysis

ADCC

Classical ADCC involves activation of NK cells by antibodies engaging toFcγRIIIA or CD16. To verify whether humanized anti-PD-1 mAbs induceADCC, NK92MI/CD16V cells, which were generated from NK92MI cells (ATCC)by co-transducing expression plasmids containing CD16 (V158 allele) andFcRγ genes, were used as effector cells, and PD-1-expressing T cellline, HuT78/PD-1, was used as target cells. NK92MI/CD16V cells (4×10⁴)were co-cultured with equal number of HuT78/PD-1 cells in 96-wellV-bottom plates for 5 h. Cytotoxicity was determined by LDH releaseassay described in previous section. The results confirmed thathu317-4B2/IgG4mt6, hu317-4B6/IgG4mt6, hu317-4B6/IgG4mt10 andhu326-4A3/IgG4mt10 all have base level of ADCC comparing to the positivecontrols (FIG. 7). The minor difference in ADCC between those 4 mAbs maybe attributable to experimental error (see error bars in FIG. 7).

CDC

Human IgG4 antibodies, in general, do not induce any CDC via classicalpathway. Whether anti-PD-1 mAbs in IgG4mt10 format will trigger CDC wasevaluated using a PD-1-expressing T cell line, Hut78/PD-1, and freshhuman serum from healthy donors. Cell lysis by CDC was determined byCelltiter glo assay kits (Promega, Beijing, China). In brief, HuT78/PD-1cells (2×10⁴) were incubated in serum-free RPMI1640 (Invitrogen) withanti-PD-1 Abs (10 μg/ml) at 37° C. for 15 minutes before adding normalhuman serum (NHS) to the final concentration of 15% or 50% in 96-wellflat-bottom plates in a total volume of 120 μl. After overnightincubation at 37° C., cells were lysed and assayed for ATPconcentration. To test whether humanized anti-PD-1 mAbs in IgG4mt10 cankill PD-1⁺ primary T cells via CDC, PBMCs isolated from healthy donorswere pre-activated with anti-CD3 Ab OKT3 (40 ng/ml) for 3 days beforeco-culture with anti-PD-1 Abs plus NHS. The amount of ATP is directlyproportional to the number of cells present in culture. Fluorescence wasread using a 96-well fluorometer (PHERA Star FS, BMG LABTECH). Theresults are expressed in relative fluoresence units (RFU) that areproportional to the number of viable cells. The percent CDC activity wascalculated as follows: % CDC activity=[RFU test−RFU background)/(RFU attotal cell lysis—RFU background)]×100. In general, we were not able todetect any ADCC mediated by anti-PD-1 mAbs in IgG4mt10 format that bindto activated PBMCs. In hypersensitive experimental conditions, such asusing PD-1 highly-expressing cell line, high serum and antibodyconcentration, we detected very low level of CDC in some occasions, andthere is not much differences between different versions and anti-PD-1mAbs, indicating the anti-PD-1 mAbs in IgG4 variant formats retained thefeature of low or no CDC activity as the common form of IgG4.

Example 10 Humanized anti-PD-1 mAbs in IgG4mt10 Format Have EnhancedStability Under Stress Conditions

Stability of Anti-PD-1 Antibodies in High Temperature and AcidicConditions

Anti-PD-1 antibodies used in stability studies were all purified fromprotein A column followed by size exclusion chromatography (SEC) asdescribed in previous sections. Following purification, the aggregatecontents of purified antibody samples were monitored in analytical sizeexclusion chromatography-high performance liquid chromatography(SEC-HPLC), which fell within the range of 0%-0.5%.

For SEC-HPLC analysis, the antibody samples were analyzed using a TSKgelG3000 SWXL column (7.8×300 mm, Cat. No. 08541, Tosoh Bioscience,Shanghai, China) under isocratic elution condition (elution buffer 0.2Msodium phosphate, pH7.2), and subsequent detection at UV-215 nm. In eachrun, 10 microliters of antibody sample was loaded onto the column andeluted at a flow rate of 1 mL/minute. The dimer or larger aggregatespecies of antibody were separated from monomeric species and thepercentages of dimers and aggregates were determined based on theintegrated peak areas from UV traces.

For speed-enhanced shelf stability study, anti-PD-1 antibodies (10-40mg/mL in PBS) were kept in incubators at 40-50° C. for 4-7 days in orderto test the stability of antibodies in high temperature condition. Theantibody samples were then analyzed for heat-induced formation of dimerand aggregates in SEC-HPLC. For each of the anti-PD-1 antibodiesanalyzed, less than 2% became higher molecular weight species (dimersand aggregates), indicating the anti-PD-1 antibodies had good stabilityin high temperature conditions.

Antibody's stability in acidic condition has been a key challenge in thedownstream manufacturing process (Liu et al. 2010 mAbs 2:480-499).Antibody elution from protein A and inactivation of virus usuallyrequire incubation of antibody in low pH (2.5-4) conditions. However,such acidic conditions could potentially cause antibody denaturation andaggregation. Human IgG4 has been known to be less stable than IgG1 andIgG2 (2002 Immunology 105:9). Therefore, we assayed the humanized mAbsmade with various IgG4 mutant forms. Briefly, Antibody stabilities inlow pH conditions were studied by 1:1 volume of each antibody sample (10mg/mL in PBS) mixed with low pH buffers containing 50 mM sodium citrate,100 mM NaCl at pH3.6, 3.3, 3.0 or 2.7, respectively. After 1 hourincubation at room temperature, the antibody samples in low pHconditions were neutralized by 1:5 dilution into SEC-HPLC elution buffercontaining 0.2M sodium phosphate, pH7.2. SEC-HPLC analyses were done asdescribed above and percentages of dimers and aggregates induced by lowpH conditions were quantified. The anti-PD-1 mAb 317-4B6 in IgG1 formatwas most stable in bioprocessing-relevant acidic conditions even when pHvalue get as low as 2.7. Among the anti-PD-1 mAbs made in several IgG4variants, hu317-4B6/IgG4mt10 and hu326-4A3/IgG4mt10 were the most stableunder the acidic buffer condition (Table 21) as the acid-inducedaggregates were significantly reduced to a level that was comparable tothat of the IgG1 format of anti-PD-1 mAbs, 317-4B6 and 326-4A3, i.e. thesoluble aggregate is less than 2% (Table 21).

TABLE 21 Dimer and soluble aggregates formed in acidic buffers andassayed by SEC-HPLC % of dimer and aggregates anti-PD-1 mAbs pH 7.2 pH3.6 pH 3.3 pH 3.0 pH 2.7 317-4B6/IgG1 0.0% 0.0% 0.2% 0.1% 0.2%317-4B6/IgG4mt1 0.0% 1.0% 11.0% 49.0%  48.0%  317-4B6/IgG4mt3 0.0% 13.0%31.0% >50%  >50%  317-4B6/IgG4mt6 0.0% 4.0% 41.0% >50%  >50% 317-4B6/IgG4mt9 0.0% 0.5% 2.1% 3.3% 2.0% 317-4B6/IgG4mt10 0.0% 0.2% 0.6%0.6% 1.4% 326-4A3/IgG4mt10 0.0% 0.0% 0.4% 0.5% 1.2%

Example 11 Mapping the Binding Epitopes of Anti-PD-1 mAbs

Previous reports about the crystal structures of PD-1/PD-L1 andPD-1/PD-L2 complexes had shed light to understanding critical amino acid(AA) residues on PD-1 which are required for the ligand-binding (Zhanget al. 2004 Immunity, 20:337-347; Lin D. Y. et al. 2008 PNAS105:3011-3016; Lazar-Molnar E. et al. 2008 PNAS, 105:10483-10488). Infact, six of such AA residues were identified on the receptor throughpoint mutation analysis required for PD-L1 binding. Five of the six AAresidues were also required for PD-L2 binding (Lin D. Y. et al. 2008PNAS 105:3011-3016). Based on the information from the structure-guidedmutation analysis we hypothesized that most effective way for functionalmAbs to block PD-1 mediated signaling is to compete with PD-1 ligands bybinding to the six critical AA residues, therefore, occupying thebinding epitopes required for the ligand binding. To explore thehypothesis and to understand the mechanism of action by functional PD-1antibodies, we have made six mutants of PD-1 by replacing each of thesix critical AAs to A1a, individually, i.e. K45A, I93A, L95A, P97A,I101A and E103A (AA residue numbering based on Lin D. Y. et al. 2008PNAS 105:3011-3016). The mutant PD-1/Fc and PD-1/His (FIG. 1) were usedas templates for PCR-guided mutagenesis or rolling-circle mutagenesisusing Fast Mutagenesis System (Cat. No. FM111, Transgen Biotech,Beijing, China). All mutants were sub-cloned in our pcDNA-basedexpression vectors, and verified by sequencing. The mutated andwild-type PD-1 proteins were expressed by transient transfection(described in Example 1), and prepared after 4 to 6 days of culture. Theconditioned media (CM) were analyzed by Western blot to verify the PD-1protein expression in terms of quality and quantity. The supernatants(CM), after clearing cell debris, were directly used in ELISA analysisor Western blot for epitope-mapping.

To study the binding epitopes of humanized anti-PD-1 mAbs, ELISA assaysusing the wild-type (WT) and mutant (Mt) PD-1 were performed to assessthe binding activities of hu317-4B5, hu317-4B6, hu326-3B1 and hu326-4A3.For comparison to check the uniqueness of the antibody bindingsignature, two reference antibody (Reference Ab-1 and Reference Ab-2from U.S. Pat. No. 8,008,449B2 and U.S. Pat. No. 8,168,757B2,respectively) were included in the study. Equal volume of CM containingWT or Mt PD-1 was coated in 96-well plate for all mAbs in the same ELISAassay. All ELISA results were normalized using the mean ELISA readingsof WT PD-1 binding signals as the standard. ELISA binding signals to aspecific Mt PD-1 were further normalized against the highest antibodybinding read-out (set as 100%) to the specific Mt PD-1. For convenienceof data analysis, When a mAb's ELISA binding signal for a specificmutant dropped below 50% relative to WT PD-1, it is defined that theamino acid residue is a significant binding epitope because whosemutation significantly abrogated the antibody binding. Likewise, if amAb's ELISA binding signal for a specific mutant dropped below 25%, itis defined as very significant. As shown in FIG. 8, two of the criticalAA residues in PD-1, K45 and I93, are significant or very significantepitopes for mAbs hu317-4B5 and hu317-4B6 binding, and three AAresidues, I93, L95 and P97, are either significant or very significantepitopes for hu326-3B1 and hu326-4A3. On the other hand, the tworeference antibodies have distinctive binding epitopes, P97 issignificant for Reference Ab-1, while L95 and P97 are significant forReference Ab-2.

Interestingly, when the PD-1 protein is denatured in Western Blot, mAbhu317-4B5 and-4B6 were still capable of binding to WT PD-1 though thecritical binding epitopes (K45 and I93) are not close to each other(non-linear). It indicated that the PD-1 protein became renatured tosome degree after denaturation in SDS-PAGE of Western Blot process,which allows the anti-PD-1 mAbs to recognize and bind to it. Taking theadvantage of this observation, we performed Western Blot analysis forall six antibodies used in above ELISA study. The overall results fromWestern Blot corroborated very well to the ELISA results, i.e. thesignificant or very significant epitopes, whose mutations resulted inlow binding signals in ELISA, also gave weakest Western Blot bandcomparing to the binding to other mutant PD-1 (FIG. 8). Some minordifferences between ELISA and Western Blot were also observed, e.g., theELISA binding signals on I93A and E103A by reference Ab-2 wererelatively stronger than those in Western Blot. It may be indicative ofthat those AA residues may also contribute to the binding because whosemutations impacted the binding though only under stress condition (i.e.denaturation or losing native conformation). As summarized in Table 22,the anti-PD-1 mAbs in this invention have identifiable binding epitopesdiffering from other anti-PD-1 antibody.

TABLE 22 Summary* of key epitopes by anti-PD-1 mAbs K45A I93A L95A P97AI101A E103A hu317-4B5 *** ** hu317-4B6 *** ** hu326-3B1 ** ** **hu326-4A3 *** ** ** Ref. Ab-1 ** Ref. Ab-2 ** ** *based on FIG. 8

Example 12 Anti-PD-1 mAbs Activate Primary Human PBMCs and Inhibit TumorGrowth in Xenograft Mouse Models

Humanized Anti-PD-1 mAbs Activate Human PBMCs

Throughout the humanization processes, the humanized anti-PD-1 mAbs atvarious stages retained similar functional activities as assessed byELISA, FACS and immune cell-based cytokine release assays. To confirmthe function of final versions of humanized mAbs, we assayed theactivating functions of hu317-4B5, hu317-4B6, hu326-3B1 and hu326-4A3using primary human PBMCs. The results demonstrated that those mAbsthroughout the humanization maintained the original murine mAb functionsto activate primary PBMCs although the degree of activation differsamong the four donors due to the variance of individual's geneticbackground (FIG. 9).

Humanized Anti-PD-1 mAbs Enhance NK Cell-Based Cytotoxicity AgainstCancer Cells

Reminiscent of the original murine mAbs, the humanized anti-PD-1 mAbs,hu317-4B5, hu317-4B6, hu326-3B1 and hu326-3G1, enhance NK92MI/PD-1cell-mediated cytotoxicity against the target lung cancer cells,SK-MES-1/PD-L1, in a dose-dependent manner (FIG. 10, Table 23). Itappeared evident that in principle the humanized anti-PD-1 mAbs mightfunction to break immune cell tolerance mediated by PD-1 signaling,enhancing the cancer killing activity by immune cells, e.g. NK cells andcytotoxic T-lymphocytes.

Humanized Anti-PD-1 mAb Activates Human PBMCs and Inhibits Tumor Growthin a Mouse Xenograft Cancer Model in Vivo

All above experimental evidences indicated that the anti-PD-1 mAbs mightwork in mouse cancer models utilizing immune-compromised micexenografted with human cancer cells, subsequently implanting human PBMCsand applying the mAb treatment to inhibit cancer cell growth in vivo.The experiment was designed as follows. Seven-eight week old SCID-malemice (Vital River Laboratories, China) were inoculated subcutaneously atright flank with 3×10⁶ Hep3B/OS8-PD-L1 cells in 50% Matrigel (BDBiosciences, New Jersey, USA). Fifteen days after tumor inoculation, themice bearing tumor size between 100-250 mm³ were randomized and dividedinto three treatment groups. One hundred microliters of pooled PBMCs(5×10⁵) from 2 healthy donors were injected intratumorally. Three dayspost PBMC-implanting, anti-PD-1 antibodies (Hu317-IgG4mt2) and human IgGwere administered via s.c. at a dose of 10 mg/kg, respectively. Antibodytreatment was repeated once every 10 days for a total of 3 times. PBSwas injected in a parallel group as negative control. Tumors weremeasured twice a week using a caliper starting on day 7. Tumor volumeswere calculated using the following formula: [D×(d²)]/2, in which Drepresents the large diameter of the tumor, and d represents the smalldiameter. All animal studies were performed following Beigene AnimalCare and Use Procedure.

In the in vivo study, although 60% of tumors in the control groups wereauto-regressed, the rest of in vivo experiment is still quiteinformative, which were presented in FIG. 11. In the control groups,either vehicle-treated or human IgG (huIgG)-treated group, each has 40%tumors (2 of 5 mice) outgrowing larger than the baseline at startingpoint. The two tumors in PBS-treated group grew much larger (above 2,000mm³, one tumor-bearing mouse was terminated earlier due to passing tumorsize limit by protocol). The two tumors in huIgG-treated group grew tothe size of 800 and 1,370 mm3, significantly above the average baselineof 164 mm³ though smaller than the PBS-treated tumors. On the otherhand, in the anti-PD-1 mAb (hu317-1/IgG4mt2)-treated group, tumors wereeither completely regressed or close to baseline size (one tumor=200mm³, which grew back slowly after regressed to 50% of baseline at twoweeks from PBMC implanting). The results indicated that the anti-PD-1mAb described above can activate human immune cells inhibiting tumorcells growth in the mouse in vivo cancer model, which is consistent tothe in vitro experimental results described above.

1-20. (canceled)
 21. An IgG monoclonal an antibody which specificallybinds human PD-1, and comprises modified hinge and γFc regions, whereinthe modified regions reduce binding of the antibody to Fc γRI,FcγRIIA-H131, FcγRIIA-R131, FcγRIIB, FcγRIIIA-F158 and FcγRIIIA-V158without reducing shelf or acid stability of the antibody.
 22. Theantibody of claim 21 wherein the antibody is an IgG1 antibody.
 23. Theantibody of claim 21 wherein the antibody is an IgG4 antibody.
 24. Theantibody of claim 21 wherein the antibody is an IgG4 antibody and themodified regions comprise a combination of mutations that is: S228P,E233P, F234V, L235A, D265A.
 25. The antibody of claim 21 wherein theantibody is an IgG4 antibody and the modified regions comprise acombination of mutations that is: S228P, E233P, F234V, L235A, D265T. 26.The antibody of claim 21 wherein the antibody is an IgG4 antibody andthe modified regions comprise a combination of mutations that is: S228P,E233P, F234V, L235A, D265A, R409K.
 27. The antibody of claim 21 whereinthe antibody is an IgG4 antibody and the modified regions comprise acombination of mutations that is: S228P, E233P, F234V, L235A, D265A,L309V, R409K.
 28. The antibody of claim 21 comprising an IgG4 heavychain effector or constant domain comprising SEQ ID NO 85 or
 86. 29. Theantibody of claim 21 comprising an IgG4 heavy chain effector or constantdomain comprising SEQ ID NO 87 or
 88. 30. A method of using an antibodyaccording to claim 21 comprising the step of administering the antibodyto a person determined to have cancer or a viral infection or tootherwise be in need of PD-1 antagonism.
 31. An IgG monoclonal antibodywhich specifically binds human PD-1, and comprises modified hinge andγFc regions, wherein the modified regions reduce ADCC and CDC effectorfunction of the antibody without reducing shelf or acid stability of theantibody.
 32. The antibody of claim 31 wherein the antibody is an IgG1antibody.
 33. The antibody of claim 31 wherein the antibody is an IgG4antibody.
 34. The antibody of claim 31 wherein the antibody is an IgG4antibody and the modified regions comprise a combination of mutationsthat is: S228P, E233P, F234V, L235A, D265A.
 35. The antibody of claim 31wherein the antibody is an IgG4 antibody and the modified regionscomprise a combination of mutations that is: S228P, E233P, F234V, L235A,D265T.
 36. The antibody of claim 31 wherein the antibody is an IgG4antibody and the modified regions comprise a combination of mutationsthat is: S228P, E233P, F234V, L235A, D265A, R409K.
 37. The antibody ofclaim 31 wherein the antibody is an IgG4 and the modified regionscomprise a combination of mutations that is: S228P, E233P, F234V, L235A,D265A, L309V, R409K.
 38. The antibody of claim 31 comprising an IgG4heavy chain effector or constant domain comprising SEQ ID NO 85 or 86.39. The antibody of claim 31 comprising IgG4 heavy chain effector orconstant domain comprising SEQ ID NO 87 or
 88. 40. A method of using anantibody according to claim 31 comprising the step of administering theantibody to a person determined to have cancer or a viral infection orto otherwise be in need of PD-1 antagonism.